Luminogenic and fluorogenic compounds and methods to detect molecules or conditions

ABSTRACT

A method to detect the presence or amount of at least one molecule in a sample which employs a derivative of luciferin or a derivative of a fluorophore is provided. Compounds and compositions for carrying out the methods of the invention are also provided.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.11/444,145, filed May 31, 2006, which application claims the benefit ofthe filing date of U.S. Application Ser. No. 60/685,957, filed May 31,2005, U.S. Application Ser. No. 60/693,034, filed Jun. 21, 2005, U.S.Application Ser. No. 60/692,925, filed Jun. 22, 2005 and U.S.Application Ser. No. 60/790,455, filed Apr. 7, 2006, the disclosures ofwhich are incorporated by reference herein.

BACKGROUND

Luminescence is produced in certain organisms as a result of aluciferase-mediated oxidation reaction. Luciferase genes from a widevariety of vastly different species, particularly the luciferase genesof Photinus pyralis and Photuris pennsylvanica (fireflies of NorthAmerica), Pyrophorus plagiophthalamus (the Jamaican click beetle),Renilla reniformis (the sea pansy), and several bacteria (e.g.,Xenorhabdus luminescens and Vibrio spp), are extremely popularluminescence reporter genes. Firefly luciferase is also a popularreporter for determining ATP concentrations, and, in that role, iswidely used to detect biomass. Luminescence is also produced by otherenzymes when those enzymes are mixed with certain synthetic substrates,for instance, alkaline phosphatase and adamantyl dioxetane phosphate, orhorseradish peroxidase and luminol.

Luciferase genes are widely used as genetic reporters due to thenon-radioactive nature, sensitivity, and extreme linear range ofluminescence assays. For instance, as few as 10⁻²⁰ moles of fireflyluciferase can be detected. Consequently, luciferase assays of geneactivity are used in virtually every experimental biological system,including both prokaryotic and eukaryotic cell cultures, transgenicplants and animals, and cell-free expression systems. Similarly,luciferase assays used to determine ATP concentration are highlysensitive, enabling detection to below 10⁻¹⁶ moles.

Luciferases can generate light via the oxidation of enzyme-specificsubstrates, e.g., luciferins. For firefly luciferase and all otherbeetle luciferases, light generation occurs in the presence ofluciferin, magnesium ions, oxygen, and ATP. For anthozoan luciferases,including Renilla luciferase, only oxygen is required along with thesubstrate coelentrazine. Generally, in luminescence assays to determinegenetic activity, reaction substrates and other luminescence activatingreagents are introduced into a biological system suspected of expressinga reporter enzyme. Resultant luminescence, if any, is then measuredusing a luminometer or any suitable radiant energy-measuring device. Theassay is very rapid and sensitive, and provides gene expression dataquickly and easily, without the need for radioactive reagents.

Because most enzymatic reactions do not generate outputs that are asideal as luciferase, the availability of a luciferase mediated assay forenzymatic reactions useful in cellular analysis and high-throughputscreening applications would be desirable to those working in thisfield. The development of such a luciferase mediated reaction as thebasis for such enzymatic or biological assays has, however, beenlimited. Luciferase mediated reactions have been employed to detectnumerous other molecules, e.g., ATP or lactate dehydrogenase. For someof those reactions, a derivative of the naturally occurring substrate isemployed. Native firefly luciferin, a polytherocyclic organic acid,D-(−)-2-(6′hydroxy-2′-benzothiazolyl)-Δ²-thiazolin-4-carbozylic acid, isshown in FIG. 1. For instance, methods for using luciferin derivativeswith a recognition site for an enzyme such as a protease as aprosubstrate were described by Miska et al. (Journal of ClinicalChemistry and Clinical Biochemistry, 25:23 (1987)). The heterogenousassays were conducted by incubating the luciferin derivative with theappropriate enzyme, e.g., a protease, for a specified period of time,then transferring an aliquot of the mixture to a solution containingluciferase. Masuda-Nishimura et al. (Letters in Applied Microbio.,30:130 (2000)) reported the use of a single tube (homogenous) assaywhich employed a galactosidase substrate-modified luciferin. In theseluciferin derivatives, the portion of the derivative functioning as thereactive group for the nonluciferase enzyme activity was coupled to theD-luciferin or aminoluciferin backbone such that upon the action of thenonluciferase enzyme, a D-luciferin or aminoluciferin molecule wasproduced as the direct product of the reaction to serve as the substratefor luciferase. A primary obstacle to broadly applying luciferasemediated reactions for other enzymatic assays has been the belief thatto modify the luciferin molecule to function as a substrate for anonluciferase enzyme, the activity of the nonluciferase enzyme mustdirectly yield a D-luciferin or aminoluciferin molecule to retain itsfunction as a substrate for luciferase.

There is, therefore, a need in the field of biological assays to expandthe utility of luciferase mediated reactions for nonluciferase enzymesby identifying derivatives of luciferin that function as a substrate fora nonluciferase enzyme or other biological molecule of interest and as aprosubstrate for luciferase regardless of whether D-luciferin oraminoluciferin is released as a direct result of the nonluciferaseenzymatic reaction.

SUMMARY OF THE INVENTION

The present invention provides derivatives of luciferin and methods forusing such derivatives in enzyme activity assays or non-enzymaticbiological assays where the luciferin derivative serves as a substratefor a desired enzyme and is a prosubstrate for luciferase or wherein theluciferin derivative is a molecule which is modified by a molecule ofinterest, which modified molecule is a substrate for luciferin.Surprisingly, many of the luciferin derivatives also have activity assubstrates for luciferase in a light generating assay. Thus, byproviding luciferin derivatives having a particular enzyme recognitionsite (reactive chemical group in a molecule referred to as a substratefor a particular enzyme) for a desired nonluciferase enzyme coupled tothe luciferin backbone (or other chemical moiety constituting a suitablesubstrate for luciferase), such as derivatives with modifications at the6′ hydroxy site of luciferin or the 6′ amino site of aminoluciferin,yielding a substrate for a desired nonluciferase enzyme and aprosubstrate of luciferase, numerous nonluciferase enzymes may bemeasured in a bioluminescent assay.

Modifications of luciferin within the scope of the derivatives of thisinvention include one or more substitutions of a ring atom, one or moresubstitutions of a substituent (atom or group) attached to a ring atom,and/or addition of one or more atoms to the ring, e.g., expansion oraddition of rings, or a combination thereof. Numbering for some of thering atoms in D-luciferin is shown in FIG. 1. Native firefly luciferinhas three linked rings, a 6 membered ring having an OH group at position6 (“ring A” or “A ring” hereinafter), a 5 membered thiazole ring linkedto the 6 membered ring (“ring B” or “B ring” hereinafter), and a 5membered thiazole ring that is modified with a carboxyl group atposition 5 (“ring C” or “C ring” hereinafter). For instance, a luciferinderivative with a A ring modification may have a substitution of a Catom in the A ring with another atom, addition of a ring, a substitutionof a substituent attached to a ring atom with a different atom or group,or any combination thereof. A luciferin derivative with a B ringmodification may have an addition to or substitution of an atom in thefive membered ring, e.g., insertion of one or more atoms, therebyexpanding the ring, for instance, to a six membered ring, substitutionof N or S in the ring with a different atom, e.g., a C or O,substitution of a substituent atom or group attached to a ring atom, orany combination thereof. A luciferin derivative with a C ringmodification may have a substitution of an atom in the ring with anotheratom, a substitution of a substituent attached to a ring atom, with adifferent atom or group, or any combination thereof. In one embodiment,a derivative of the invention is one which is modified at more than oneposition, for instance, the derivative has two (or more) A ringmodifications, two (or more) B ring modifications, two (or more) C ringmodifications, or any combination thereof. In one embodiment, onemodification is the substitution of a substituent on one of the rings ofD-luciferin with a substrate for a nonluciferase enzyme, or a linker anda substrate for the nonluciferase enzyme.

Exemplary derivatives with A ring modifications may be a substrate for areductase, such as a cytochrome P450 reductase, monoamine oxidase (MAO),flavin monooxygenase (FMO), glutathione S transferase (GST), dealkylase,deacetylase, deformylase, phosphatase, e.g., alkaline phosphatase (AP),sulfatase, beta-lactamase, alcohol dehydrogenase, protease e.g.,proteosome, cathepsin, calpain, beta secretase, thrombin, or granzyme,luciferase, or useful to detect reactive oxygen species (ROS),peroxidase, e.g., horseradish peroxidase (HRP), and redox conditions.Exemplary molecules or conditions to be detected with derivatives havingat least a B ring modification include but are not limited todealkylase, GST or luciferase, or redox conditions. Exemplary moleculesto be detected with those derivatives include a cytochrome P450 enzyme,esterase, e.g., acetylcholinesterase, OH radicals, demethylase,deacetylase, deformylase, or mycoplasma carboxypeptidase. Exemplarymolecules to be detected with derivatives having C ring modificationsinclude but are not limited to esterases.

In one embodiment, derivatives of luciferin or aminoluciferin have thefollowing structure: L-X-M-Y—R (compound of formula IV), wherein L, ifpresent, may be a substrate for an enzyme or another molecule whichinteracts with the enzyme; X may be O, NH, or a linker, e.g., aself-cleavable linker which spontaneously cleaves to yield M-Y—R after Lhas been removed from L-X-M-Y—R; M may be luciferin, quinolinylluciferin or naphthyl luciferin, or aminoluciferin or aminoquinolinylluciferin, Y is O (ester), NH (amide), NH—NH (hydrazide), or S(thioester); and R, if present, may be alkyl, an aromatic molecule, apeptide, an oligonucleotide, or a self-cleavable linker attached to asubstrate for an enzyme.

In one embodiment, the invention provides a compound of formula I:

wherein

Y is N,N-oxide, N—(C₁-C₆)alkyl, or CH;

when Y is N, then X is not S;

X is S, O, CH═CH, N═CH, or CH═N;

when X is S, then Y is not N;

Z and Z′ are independently H, OR, NHR, or NRR;

Z″ is O, S, NH, NHR, or N═N;

Q is carbonyl or CH₂;

W¹ is H, halo, (C₁-C₆)alkyl, (C₂-C₂₀)alkenyl, hydroxyl, or(C₁-C₆)alkoxy; or

W¹ and Z are both keto groups on ring A, and at least one of the dottedlines denoting optional double bonds in ring A is absent;

each W² is independently H, halo, (C₁-C₆)alkyl, (C₂-C₄)alkenyl,hydroxyl, or (C₁-C₆)alkoxy;

each of K¹, K², K³, and K⁴ are independently CH, N,N-oxide, orN—(C₁-C₆)alkyl, and the dotted lines between K¹ and K², and K³ and K⁴,denote optional double bonds;

A′ and B′ are optional aromatic rings fused to ring A, only one of whichis present in the compound, so as to form a fused tricyclic system; and

-   -   when B′ is present, the group Z is present, and    -   when A′ is present, the group Z is absent; and

the dotted line in ring B is an optional double bond;

each R is independently H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₁₂)alkoxy, (C₆-C₃₀)aryl,heteroaryl, heterocycle, (C₁-C₂₀)alkylsulfoxy, (C₆-C₃₀)arylsulfoxy,heteroarylsulfoxy, (C₁-C₂₀)alkylsulfonyl, (C₆-C₃₀)arylsulfonyl,heteroarylsulfonyl, (C₁-C₂₀)alkylsulfinyl, (C₆-C₃₀)arylsulfinyl,heteroarylsulfinyl, (C₁-C₂₀)alkoxycarbonyl, amino, NH(C₁-C₆)alkyl,N((C₁-C₆)alkyl)₂, tri(C₁-C₂₀)ammonium(C₁-C₂₀)alkyl,heteroaryl(C₁-C₂₀)alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen, (C₆-C₃₀)arylthio,(C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, (C₆-C₃₀)arylphosphate,(C₆-C₃₀)arylphosphonate, phosphate, sulfate, saccharide, or M⁺optionally when Z″ is oxygen, wherein M is an alkali metal;

or when Z or Z′ is NR¹R¹, R¹R¹ together with the N to which they areattached forms a heteroaryl or heterocycle group;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, amino, aryl,heteroaryl, or heterocycle group is optionally substituted with 1, 2, 3,4, or 5 substituents selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxyl,(C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —COOR^(x),—SO₂R^(x), —SO₃R^(x), nitro, amino, (C₁-C₂₀)alkyl-S(O)—,(C₁-C₂₀)alkyl-SO₂—, phosphate, (C₁-C₂₀)alkylphosphate,(C₁-C₂₀)alkylphosphonate, NH(C₁-C₆)alkyl, NH(C₁-C₆)alkynyl,N((C₁-C₆)alkyl)₂, N((C₁-C₆)alkynyl)₂, mercapto, (C₁-C₂₀)alkylthio,(C₆-C₃₀)aryl, (C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, andheterocycle, and each substituent is optionally substituted with one tothree R groups;

R^(x) is H, (C₁-C₆)alkyl, or (C₆-C₃₀)aryl;

when Z or Z′ comprises a nitrogen moiety, one or both of the hydrogensof the Z or Z′ nitrogen moiety may be replaced by (C₁-C₂₀)alkyl or thegroup L, wherein L is an amino acid radical, a peptide radical having upto 20 amino acid moieties, or any other small molecule that is asubstrate for a nonluciferase; with the proviso that when L is an aminoacid radical or a peptide radical, at least one W² is not H;

when Z is a hydroxyl group or a nitrogen moiety, H of the hydroxyl ornitrogen moiety may be replaced by (HO)₂P(O)—OCH₂—, sulfo, —PO₃H₂, or bya cephalosporanic acid attached to the group Z via a carbon chain of oneto about 12 carbon atoms; with the proviso that when ring B is athiazole ring, the sulfo or the —PO₃H₂ group is attached to the hydroxyloxygen via a (C₁-C₆)alkylene group;

when Z or Z′ is a hydroxyl group or a nitrogen moiety, or when Z″—R is ahydroxyl group, one H of the hydroxyl or nitrogen moiety may be replacedby the group L′-linker, wherein L′ is a group removable by an enzyme tofree the linker, and linker is a carbon chain that can self-cleave,optionally interrupted by one or more nitrogen atoms, oxygen atoms,carbonyl groups, optionally substituted aromatic rings, or peptidebonds,

linker is attached to L′ via an oxygen atom or an NH group at oneterminus of the linker and the other terminus of the linker forms anether, ester, or amide linkage with a group Z, Z′, or Z″—R;

when Z is OR, formula I is optionally a dimer connected at the two Arings via a linker comprising a (C₁-C₁₂)alkyl diradical that isoptionally interrupted by one to four O atoms, N atoms, or an optionallysubstituted aryl, heteroaryl, or heterocycle group to form a bridgebetween the dimer of formula I, and the R group of each Z groupconnecting the dimer of formula I is replaced by the bridge;

A⁻ is an anion, present when a quaternary nitrogen is present;

or a salt thereof;

provided that:

when rings A and B form a naphthalene or quinoline ring system, then W¹is not hydrogen;

when a ring A substituent is OH, then -Q-Z″—R is not —C(O)—NH—NH₂;

when Y is N or CH and X is CH═CH and W¹ is H, then Z is not OH attachedto K³; and

when Y is N or CH and X is CH═CH and Z is H, then W¹ is not OH attachedto K³.

In another embodiment, the invention provides a compound of formula IA:

wherein

Y is N,N-oxide, N—(C₁-C₆)alkyl, or CH;

when Y is N, then X is not S;

X is S, O, CH═CH, N═CH, or CH═N;

when X is S, then Y is not N;

Z is H, OR, NHR, or NRR;

Z″ is O, S, NH, NHR, or N═N;

W¹ is H, halo, hydroxyl, (C₁-C₆)alkyl, or (C₁-C₆)alkoxy;

the dotted line in ring B is an optional double bond;

each R is independently H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₁₂)alkoxy, (C₆-C₃₀)aryl,heteroaryl, heterocycle, (C₁-C₂₀)alkylsulfoxy, (C₆-C₃₀)arylsulfoxy,heteroarylsulfoxy, (C₁-C₂₀)alkylsulfonyl, (C₆-C₃₀)arylsulfonyl,heteroarylsulfonyl, (C₁-C₂₀)alkylsulfinyl, (C₆-C₃₀)arylsulfinyl,heteroarylsulfinyl, (C₁-C₂₀)alkoxycarbonyl, amino, NH(C₁-C₆)alkyl,N((C₁-C₆)alkyl)₂, tri(C₁-C₂₀)ammonium(C₁-C₂₀)alkyl,heteroaryl(C₁-C₂₀)alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen, (C₆-C₃₀)arylthio,(C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, (C₆-C₃₀)arylphosphate,(C₆-C₃₀)arylphosphonate, phosphate, sulfate, saccharide, or M⁺optionally when Z″ is oxygen, wherein M is an alkali metal;

or when Z or Z′ is NR¹R¹, R¹R¹ together with the N to which they areattached forms a heteroaryl or heterocycle group;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, amino, aryl,heteroaryl, or heterocycle group is optionally substituted with 1, 2, 3,4, or 5 substituents selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxyl,(C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —COOR^(x),—SO₂R^(x), —SO₃R^(x), nitro, amino, (C₁-C₂₀)alkyl-S(O)—,(C₁-C₂₀)alkyl-SO₂—, phosphate, (C₁-C₂₀)alkylphosphate,(C₁-C₂₀)alkylphosphonate, NH(C₁-C₆)alkyl, NH(C₁-C₆)alkynyl,N((C₁-C₆)alkyl)₂, N((C₁-C₆)alkynyl)₂, mercapto, (C₁-C₂₀)alkylthio,(C₆-C₃₀)aryl, (C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, andheterocycle, and each substituent is optionally substituted with one tothree R groups;

R^(x) is H, (C₁-C₆)alkyl, or (C₆-C₃₀)aryl;

A⁻ is an anion, present when a quaternary nitrogen is present;

or a salt thereof;

provided that:

when rings A and B form a naphthalene or quinoline ring system, then W¹is not hydrogen;

when a ring A substituent is OH, then -Q-Z″—R is not —C(O)—NH—NH₂;

when Y is N or CH and X is CH═CH and W¹ is H, then Z is not OH attachedto carbon-6 of ring A (carbon 6 as shown in FIG. 1); and

when Y is N or CH and X is CH═CH and Z is H, then W¹ is not OH attachedto carbon-6 of ring A.

In another embodiment, the invention provides a compound of formula II:

wherein

Z and Z′ are independently OR¹, NHR¹, or NR¹R¹;

Z″ is O, S, NH, NHR, or N═N;

Q is carbonyl or CH₂;

W¹ is H, halo, (C₁-C₆)alkyl, (C₂-C₂₀)alkenyl, hydroxyl, or(C₁-C₆)alkoxy; or

W¹ and Z are both keto groups on ring A, and at least one of the dottedlines denoting optional double bonds in ring A is absent;

each W² is independently H, halo, (C₁-C₆)alkyl, (C₂-C₄)alkenyl,hydroxyl, or (C₁-C₆)alkoxy;

each of K¹, K², K³, and K⁴ are independently CH, N,N-oxide, orN—(C₁-C₆)alkyl, and the dotted lines between K¹ and K², and K³ and K⁴,denote optional double bonds;

A′ and B′ are optional aromatic rings fused to ring A, only one of whichis present in the compound, so as to form a fused tricyclic system; and

-   -   when B′ is present, the group Z is present, and    -   when A′ is present, the group Z is absent; and

R is H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₃-C₂₀)cycloalkyl, (C₁-C₁₂)alkoxy, (C₆-C₃₀)aryl, heteroaryl,heterocycle, (C₁-C₂₀)alkylsulfoxy, (C₆-C₃₀)arylsulfoxy,heteroarylsulfoxy, (C₁-C₂₀)alkoxycarbonyl, amino, NH(C₁-C₆)alkyl,N((C₁-C₆)alkyl)₂, tri(C₁-C₂₀)ammonium(C₁-C₂₀)alkyl,heteroaryl(C₁-C₂₀)alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen, saccharide, or M⁺optionally when Z″ is oxygen, wherein M is an alkali metal;

R¹ is (C₆-C₃₀)aryl, heteroaryl, heterocycle, (C₁-C₂₀)alkylthio,(C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂, —SO₃(C₁-C₂₀)alkyl, saccharide,(C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, (C₆-C₃₀)arylthio,(C₆-C₃₀)aryl-S(O)—, (C₆-C₃₀)aryl-SO₂, —SO₃(C₆-C₃₀)aryl,(C₆-C₃₀)arylphosphate, (C₆-C₃₀)arylphosphonate, or R¹ is (C₁-C₂₀)alkylsubstituted by R²;

R² is (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxyl, (C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, hydroxyl,—COOR^(x), —SO₃R^(x), (C₁-C₂₀)alkylthio, (C₆-C₃₀)arylthio,(C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂—, nitro, amino, NH(C₁-C₆)alkyl,NH(C₁-C₆)alkynyl, N((C₁-C₆)alkyl)₂, or N((C₁-C₆)alkynyl)₂, mercapto,saccharide, or trifluoromethyl;

or when Z or Z′ is NR¹R¹, R¹R¹ together with the N to which they areattached forms a heteroaryl or heterocycle group;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, amino, aryl,heteroaryl, or heterocycle group is optionally substituted with 1, 2, 3,4, or 5 substituents selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxyl,(C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —COOR^(x),—SO₂R^(x), —SO₃R^(x), (C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂—,phosphate, (C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, nitro,amino, NH(C₁-C₆)alkyl, NH(C₁-C₆)alkynyl, N((C₁-C₆)alkyl)₂,N((C₁-C₆)alkynyl)₂, mercapto, (C₁-C₂₀)alkylthio, (C₆-C₃₀)aryl,(C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, and heterocycle, andeach substituent is optionally substituted with one to three R groups;

R^(x) is H or (C₁-C₆)alkyl;

when Z or Z′ comprises a nitrogen moiety, a hydrogen of the Z or Z′nitrogen moiety may be replaced by the group L, wherein L is an aminoacid radical, a peptide radical having up to 20 amino acid moieties, orany other small molecule that is a substrate for a nonluciferase; withthe proviso that when L is an amino acid radical or a peptide radical,at least one of W¹ or a W² is not H;

when Z is a hydroxyl group or a nitrogen moiety, H of the hydroxyl ornitrogen moiety may be replaced by (HO)₂P(O)—OCH₂—, sulfo, —PO₃H₂, or bya cephalosporanic acid attached to the group Z via a carbon chain of oneto about 12 carbon atoms; with the proviso that the sulfo or the —PO₃H₂group is attached to the hydroxyl oxygen via a (C₁-C₆)alkylene group;

when Z or Z′ is a hydroxyl group or a nitrogen moiety, or when Z″—R is ahydroxyl group, one H of the hydroxyl or nitrogen moiety may be replacedby the group L′-linker, wherein L′ is a group removable by an enzyme tofree the linker, and linker is a carbon chain that can self-cleave,optionally interrupted by one or more nitrogen atoms, oxygen atoms,carbonyl groups, optionally substituted aromatic rings, or peptidebonds,

linker is attached to L′ via an oxygen atom or an NH group at oneterminus of the linker and the other terminus of the linker forms anether, ester, or amide linkage with a group Z, Z′, or Z″—R;

when Z is OR¹, formula II is optionally a dimer connected at the two Arings via linker comprising a (C₁-C₁₂)alkyl diradical that is optionallyinterrupted by one to four O atoms, N atoms, or an optionallysubstituted aryl, heteroaryl, or heterocycle group to form a bridgebetween the dimer of formula II, and the R¹ group of each Z groupconnecting the dimer of formula II is replaced by the bridge;

provided that a saccharide is not directly attached to K³;

A⁻ is an anion, present when a quaternary nitrogen is present;

or a salt thereof.

In yet another embodiment, the invention provides a compound of formulaIIA:

wherein

Z is OR¹, NHR¹, or NR¹R¹;

Z″ is O, S, NH, NHR, or N═N;

W¹ is H, halo, hydroxyl, (C₁-C₆)alkyl, or (C₁-C₆)alkoxy;

R is H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₃-C₂₀)cycloalkyl, (C₁-C₁₂)alkoxy, (C₆-C₃₀)aryl, heteroaryl,heterocycle, (C₁-C₂₀)alkylsulfoxy, (C₆-C₃₀)arylsulfoxy,heteroarylsulfoxy, (C₁-C₂₀)alkoxycarbonyl, amino, NH(C₁-C₆)alkyl,N((C₁-C₆)alkyl)₂, tri(C₁-C₂₀)ammonium(C₁-C₂₀)alkyl,heteroaryl(C₁-C₂₀)alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen, saccharide, or M⁺optionally when Z″ is oxygen, wherein M is an alkali metal;

R¹ is (C₆-C₃₀)aryl, heteroaryl, heterocycle, (C₁-C₂₀)alkylthio,(C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂, —SO₃(C₁-C₂₀)alkyl, saccharide,(C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, (C₆-C₃₀)arylthio,(C₆-C₃₀)aryl-S(O)—, (C₆-C₃₀)aryl-SO₂, —SO₃(C₆-C₃₀)aryl,(C₆-C₃₀)arylphosphate, (C₆-C₃₀)arylphosphonate, or R¹ is (C₁-C₂₀)alkylsubstituted by R²;

R² is (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxyl, (C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, hydroxyl,—COOR^(x), —SO₃R^(x), (C₁-C₂₀)alkylthio, (C₆-C₃₀)arylthio,(C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂—, nitro, amino, NH(C₁-C₆)alkyl,NH(C₁-C₆)alkynyl, N((C₁-C₆)alkyl)₂, or N((C₁-C₆)alkynyl)₂, mercapto,saccharide, or trifluoromethyl;

or when Z or Z′ is NR¹R¹, R¹R¹ together with the N to which they areattached forms a heteroaryl or heterocycle group;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, amino, aryl,heteroaryl, or heterocycle group is optionally substituted with 1, 2, 3,4, or 5 substituents selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxyl,(C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —COOR^(x),—SO₂R^(x), —SO₃R^(x), (C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂—,phosphate, (C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, nitro,amino, NH(C₁-C₆)alkyl, NH(C₁-C₆)alkynyl, N((C₁-C₆)alkyl)₂,N((C₁-C₆)alkynyl)₂, mercapto, (C₁-C₂₀)alkylthio, (C₆-C₃₀)aryl,(C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, and heterocycle, andeach substituent is optionally substituted with one to three R groups;

R^(x) is H or (C₁-C₆)alkyl;

when Z is OR¹, formula IIA is optionally a dimer connected at the two Arings via linker comprising a (C₁-C₁₂)alkyl diradical that is optionallyinterrupted by one to four O atoms, N atoms, or an optionallysubstituted aryl, heteroaryl, or heterocycle group to form a bridgebetween the dimer of formula IIA, and the R¹ group of each Z groupconnecting the dimer of formula II is replaced by the bridge;

provided that a saccharide is not directly attached to K³;

A⁻ is an anion, present when a quaternary nitrogen is present;

or a salt thereof.

Other deriviates and their use in luminogenic assays is describedhereinbelow.

The use of the luciferin derivatives described herein can result in anassay which produces a measurable change in optical properties uponinteraction with a nonluciferase molecule, which interaction may alterthe structure of the luciferin derivative. As described herein, theproduct of a reaction between a luciferin derivative and a nonluciferaseenzyme or other molecule of interest need not be D-luciferin oraminoluciferin. For example, a luciferin derivative may include asubstrate that includes a reactive chemical group for a nonluciferaseenzyme linked to luciferin or aminoluciferin via a chemical linker.Transformation of the reactive chemical group of the derivative by thenonluciferase enzyme may yield a product that contains (retains) aportion of the substrate, a portion of the chemical linker, the chemicallinker, or a portion of the substrate and the chemical linker, and thatproduct is a substrate for luciferase. Also provided are luciferinderivatives which, after interaction with a nonluciferase enzyme orother molecule, may yield a product that optionally undergoes one ormore further reactions, e.g., O-elimination, to yield a suitablesubstrate for luciferase. Luciferin derivatives in which the backbone ofluciferin is further modified in its ring structure, e.g., a quinolyl ornapthyl luciferin, are provided, as well as advantageously providingmodifications at the carboxy position of the thiazole ring, to provideimproved characteristics to the luciferin derivative. Derivatives withcertain modifications provide for or improve assays for certainnonluciferase enzymes or molecules. For instance, as describedhereinbelow, a pH insensitive derivative of luciferin was identifiedthat is useful in biological assays that may be run at a pH other thanphysiological pH, i.e., less than about pH 7.0 and greater than about pH7.8. Thus, bioluminescent methods that employ a luciferin derivative ofthe invention may be used to detect one or more molecules, e.g., anenzyme, a cofactor for an enzymatic reaction such as ATP, an enzymesubstrate, an enzyme inhibitor, an enzyme activator, or OH radicals, orone or more conditions, e.g., redox conditions.

In one embodiment, the methods employ a compound of formula III:

wherein

Y is N,N-oxide, N—(C₁-C₆)alkyl, or CH;

X is S, O, CH═CH, N═CH, or CH═N;

Z and Z′ are independently H, OR, NHR, or NRR; Z″ is O, S, NH, NHR, orN═N;

Q is carbonyl or CH₂;

W¹ is H, halo, (C₁-C₆)alkyl, (C₂-C₂₀)alkenyl, hydroxyl, or(C₁-C₆)alkoxy; or

W¹ and Z are both keto groups on ring A, and at least one of the dottedlines denoting optional double bonds in ring A is absent;

each W² is independently H, halo, (C₁-C₆)alkyl, (C₂-C₄)alkenyl,hydroxyl, or (C₁-C₆)alkoxy;

each of K¹, K², K³, and K⁴ are independently CH, N,N-oxide, orN—(C₁-C₆)alkyl, and the dotted lines between K¹ and K², and K³ and K⁴,denote optional double bonds;

A′ and B′ are optional aromatic rings fused to ring A, only one of whichis present in the compound, so as to form a fused tricyclic system; and

-   -   when B′ is present, the group Z is present, and    -   when A′ is present, the group Z is absent; and

the dotted line in ring B is an optional double bond;

each R is independently H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₁₂)alkoxy, (C₆-C₃₀)aryl,heteroaryl, heterocycle, (C₁-C₂₀)alkylsulfoxy, (C₆-C₃₀)arylsulfoxy,heteroarylsulfoxy, (C₁-C₂₀)alkylsulfonyl, (C₆-C₃₀)arylsulfonyl,heteroarylsulfonyl, (C₁-C₂₀)alkylsulfinyl, (C₆-C₃₀)arylsulfinyl,heteroarylsulfinyl, (C₁-C₂₀)alkoxycarbonyl, amino, NH(C₁-C₆)alkyl,N((C₁-C₆)alkyl)₂, tri(C₁-C₂₀)ammonium(C₁-C₂₀)alkyl,heteroaryl(C₁-C₂₀)alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen, (C₆-C₃₀)arylthio,(C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, (C₆-C₃₀)arylphosphate,(C₆-C₃₀)arylphosphonate, phosphate, sulfate, saccharide, or M⁺optionally when Z″ is oxygen, wherein M is an alkali metal;

or when Z or Z′ is NR¹R¹, R¹R¹ together with the N to which they areattached forms a heteroaryl or heterocycle group;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, amino, aryl,heteroaryl, or heterocycle group is optionally substituted with 1, 2, 3,4, or 5 substituents selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxyl,(C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —COOR^(x),—SO₂R^(x), —SO₃R^(x), nitro, amino, (C₁-C₂₀)alkyl-S(O)—,(C₁-C₂₀)alkyl-SO₂—, phosphate, (C₁-C₂₀)alkylphosphate,(C₁-C₂₀)alkylphosphonate, NH(C₁-C₆)alkyl, NH(C₁-C₆)alkynyl,N((C₁-C₆)alkyl)₂, N((C₁-C₆)alkynyl)₂, mercapto, (C₁-C₂₀)alkylthio,(C₆-C₃₀)aryl, (C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, andheterocycle, and each substituent is optionally substituted with one tothree R groups;

R^(x) is H, (C₁-C₆)alkyl, or (C₆-C₃₀)aryl;

when Z or Z′ comprises a nitrogen moiety, one or both of the hydrogensof the Z or Z′ nitrogen moiety may be replaced by (C₁-C₂₀)alkyl or thegroup L, wherein L is an amino acid radical, a peptide radical having upto 20 amino acid moieties, or any other small molecule that is asubstrate for a nonluciferase; with the proviso that when L is an aminoacid radical or a peptide radical, at least one W² is not H;

when Z is a hydroxyl group or a nitrogen moiety, H of the hydroxyl ornitrogen moiety may be replaced by (HO)₂P(O)—OCH₂—, sulfo, —PO₃H₂, or bya cephalosporanic acid attached to the group Z via a carbon chain of oneto about 12 carbon atoms; with the proviso that when ring B is athiazole ring, the sulfo or the —PO₃H₂ group is attached to the hydroxyloxygen via a (C₁-C₆)alkylene group;

when Z or Z′ is a hydroxyl group or a nitrogen moiety, or when Z″—R is ahydroxyl group, one H of the hydroxyl or nitrogen moiety may be replacedby the group L′-linker, wherein L′ is a group removable by an enzyme tofree the linker, and linker is a carbon chain that can self-cleave,optionally interrupted by one or more nitrogen atoms, oxygen atoms,carbonyl groups, optionally substituted aromatic rings, or peptidebonds,

linker is attached to L′ via an oxygen atom or an NH group at oneterminus of the linker and the other terminus of the linker forms anether, ester, or amide linkage with a group Z, Z′, or Z″—R;

when Z is OR, formula III is optionally a dimer connected at the two Arings via a linker comprising a (C₁-C₁₂)alkyl diradical that isoptionally interrupted by one to four O atoms, N atoms, or an optionallysubstituted aryl, heteroaryl, or heterocycle group to form a bridgebetween the dimer of formula III, and the R group of each Z groupconnecting the dimer of formula III is replaced by the bridge;

A⁻ is an anion, present when a quaternary nitrogen is present;

or a salt thereof;

provided that the compound of formula III is not aminoluciferin that hasbeen modified to include a protease substrate via a peptide bond at theamino group which optionally also has a protected carboxyl group atposition 5 on ring C, or any of luciferin 6′-methyl ether, luciferin6′-chloroethyl ether, 6′-deoxyluciferin, 6′-luciferin-4-trifluoromethylbenzylether, luciferin 6′-phenylethylether, luciferin 6′-geranyl ether,luciferin 6′-ethyl ether, 6′-luciferin prenyl ether, 6′-luciferin2-picolinylether, 6′-luciferin 3-picolinylether, 6′-luciferin4-picolinyl ether, luciferin 6′-benzyl ether,D-luciferin-O-β-galacto-pyranoside, D-luciferin-O-sulfate,D-luciferin-O-phosphate, D-luciferyl-L-phenylalanine, andD-luciferyl-L-N^(α)-arginine; or not a substrate for one or morecytochrome P450 enzymes but optionally may be a substrate for anon-cytochrome P450 enzyme.

In one embodiment, a bioluminescent assay method to detect one or morenonluciferase enzymes is provided. The method includes contacting asample suspected of having one or more nonluciferase enzymes, asubstrate or a co-factor for the reaction, with a corresponding reactionmixture that includes a derivative of luciferin or a derivative ofaminoluciferin that is a substrate for the nonluciferase enzyme. In oneembodiment, the derivative is one having a modification in the benzenering of D-luciferin that includes a recognition site for thenonluciferase enzyme, e.g., for a monoamine oxidase. In anotherembodiment, the derivative is one having a modification in the thiazolering (ring B) of D-luciferin which derivative is a substrate forluciferase and optionally a substrate for a nonluciferase enzyme. Inanother embodiment, the derivative is one having a modification in thethiazole ring (ring C) of luciferin that includes a recognition site foran enzyme of interest, e.g., acetylcholinesterase. In anotherembodiment, the derivative is one having a modification to one of therings that includes a recognition site for the enzyme of interest, aswell as a further modification to that ring or one or more of the otherrings.

Previously, enzymes that could be tested with luciferin derivatives werethose that interacted well close to aromatic structures (D-luciferin oraminoluciferin) and interacted with a recognition site (substrate) thatwas stable close to aromatic structures. As described herein, luciferinderivatives that are substrates for nonluciferase enzymes that do notnecessarily react with structures close to aromatic rings or those withrecognition sites that are more stable when attached to an aryl chainthan an aromatic structure, were identified. For example, an assay whichemployed previously described luciferin derivatives with a phosphategroup attached through the hydroxyl group on the benzene ring, i.e., asubstrate for alkaline phosphatase, was limited by high backgroundbecause the phosphate spontaneously hydrolyzed. Attaching the phosphateto an aryl chain is likely stabilizing, which may allow for a derivativethat is a substrate for alkaline phosphatase and may reduce thebackground. Thus, the sensitivity and utility of a phosphatase assaysuch as an alkaline phosphatase assay is increased employing a luciferinderivative of the invention.

Generally, luciferase substrates have a free hydroxyl (luciferin) orfree amino group (amino luciferin) on the benzene ring. Alternatebackbones for luciferin, like quinolinyl luciferin or napthyl luciferin,with free hydroxyl or free amino groups, are luciferase substrates. Toexpand the scaffolding upon which modifications can be made to luciferinand result in a luciferase substrate, luciferin derivatives with aryl orother chains attached through the oxygen or nitrogen on the A ring ofluciferin were prepared. Derivatives with nitrogen on the A ring wereutilized by beetle luciferase to generate bright luminescence. Moreover,HPLC verified that luciferin derivatives with aryl or alkyl chainsdecreased in concentration when acted upon by a thermostable luciferase(as opposed to wild-type luciferin contamination causing the light anddecreasing in concentration). Such luciferin derivatives are also shownherein to be utilized by other luciferases. Moreover, those scaffolds,e.g., luciferase substrates that have groups attached through the oxygenor nitrogen on the A ring of luciferin, may be modified to include asubstrate for an enzyme, a binding site for another molecule, or anyreactive group useful to measure a molecule such as a cellular bioactivemolecule, including second messengers, e.g., a cAMP binding site, forinstance, to measure cAMP, calmodulin, e.g., to measure calcium, or tomeasure IP3.

In one embodiment, a method to detect luciferase employs a compound offormula IIIA:

wherein

Y is N,N-oxide, N—(C₁-C₆)alkyl, or CH;

X is S, O, CH═CH, N═CH, or CH═N;

Z is H, OR, NHR, or NRR;

Z″ is O, S, NH, NHR, or N═N;

W¹ is H, halo, hydroxyl, (C₁-C₆)alkyl, (C₂-C₁₀)alkenyl, or(C₁-C₆)alkoxy;

W² is H, F, or methyl;

the dotted line in ring B is an optional double bond;

each R is independently H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₁₂)alkoxy, (C₆-C₃₀)aryl,heteroaryl, heterocycle, (C₁-C₂₀)alkylsulfoxy, (C₆-C₃₀)arylsulfoxy,heteroarylsulfoxy, (C₁-C₂₀)alkylsulfonyl, (C₆-C₃₀)arylsulfonyl,heteroarylsulfonyl, (C₁-C₂₀)alkylsulfinyl, (C₆-C₃₀)arylsulfinyl,heteroarylsulfinyl, (C₁-C₂₀)alkoxycarbonyl, amino, NH(C₁-C₆)alkyl,N((C₁-C₆)alkyl)₂, tri(C₁-C₂₀)ammonium(C₁-C₂₀)alkyl,heteroaryl(C₁-C₂₀)alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen, (C₆-C₃₀)arylthio,(C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, (C₆-C₃₀)arylphosphate,(C₆-C₃₀)arylphosphonate, phosphate, sulfate, saccharide, or M⁺optionally when Z″ is oxygen, wherein M is an alkali metal;

or when Z or Z′ is NR¹R¹, R¹R¹ together with the N to which they areattached forms a heteroaryl or heterocycle group;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, amino, aryl,heteroaryl, or heterocycle group is optionally substituted with 1, 2, 3,4, or 5 substituents selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxyl,(C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —COOR^(x),—SO₂R^(x), —SO₃R^(x), nitro, amino, (C₁-C₂₀)alkyl-S(O)—,(C₁-C₂₀)alkyl-SO₂—, phosphate, (C₁-C₂₀)alkylphosphate,(C₁-C₂₀)alkylphosphonate, NH(C₁-C₆)alkyl, NH(C₁-C₆)alkynyl,N((C₁-C₆)alkyl)₂, N((C₁-C₆)alkynyl)₂, mercapto, (C₁-C₂₀)alkylthio,(C₆-C₃₀)aryl, (C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, andheterocycle, and each substituent is optionally substituted with one tothree R groups;

R^(x) is H, (C₁-C₆)alkyl, or (C₆-C₃₀)aryl;

when Z is OR, formula III is optionally a dimer connected at the two Arings via a linker comprising a (C₁-C₁₂)alkyl diradical that isoptionally interrupted by one to four O atoms, N atoms, an optionallysubstituted aryl, heteroaryl, or heterocycle group, or a combinationthereof, to form a bridge between the dimer of formula III, and the Rgroup of each Z group connecting the dimer of formula III is replaced bythe bridge;

A⁻ is an anion, present when a quaternary nitrogen is present;

or salt thereof;

provided that the compound of formula III is not aminoluciferin that hasbeen modified to include a protease substrate via a peptide bond at theamino group which optionally also has a protected carboxyl group atposition 5 on ring C, or any of luciferin 6′-methyl ether, luciferin6′-chloroethyl ether, 6′-deoxyluciferin, 6′-luciferin-4-trifluoromethylbenzylether, luciferin 6′-phenylethylether, luciferin 6′-geranyl ether,luciferin 6′-ethyl ether, 6′-luciferin prenyl ether, 6′-luciferin2-picolinylether, 6′-luciferin 3-picolinylether, 6′-luciferin4-picolinyl ether, luciferin 6′-benzyl ether,D-luciferin-O-β-galacto-pyranoside, D-luciferin-O-sulfate,D-luciferin-O-phosphate, D-luciferyl-L-phenylalanine, andD-luciferyl-L-N^(α)-arginine; or not a substrate for one or morecytochrome P450 enzymes but optionally may be a substrate for anon-cytochrome P450 enzyme.

Furthermore, the inclusion of a thiol compound with a luciferinderivative in a luciferase-mediated assay may effectively stabilize theluminescence of the reaction, thereby providing for “glow” kinetics,i.e., luminescent intensity of the luciferase-mediated reaction isrelatively constant over time after addition of the derivative. Suchcompounds may be also be used in bioluminogenic assays monitoring thepresence or activity of nonluciferase enzymes or a nonenzymaticbiological reaction.

For derivatives that function directly as a substrate for luciferase, aswell as optionally a substrate of a nonluciferase enzyme or othermolecules, the derivative may be employed to directly detect luciferase,or a co-factor, inhibitor, or activator of the luciferase reaction. Ifthe derivative is a prosubstrate for luciferase, i.e., the product of areaction between the derivative and the nonluciferase enzyme is asubstrate for luciferase, sequential or concurrent reactions for thenonluciferase enzyme and the luciferase may be conducted. For instance,an assay for a nonluciferase enzyme that includes a luciferin derivativethat is a prosubstrate for luciferase may be conducted in a singlereaction vessel and a beetle luciferase reaction mixture added to thatvessel. In another embodiment, a reaction mixture for an assay for anonluciferase enzyme that includes a luciferin derivative that is aprosubstrate for luciferin may be conducted in a single reaction vesseland a portion of that reaction added to a different vessel having abeetle luciferase reaction mixture. Alternatively, the nonluciferase andluciferase reactions may be conducted simultaneously in the same vessel.

The invention thus provides in an embodiment a method to detect ordetermine the presence or amount of a molecule for a nonluciferaseenzyme-mediated reaction in a sample. The method includes contacting asample, a first reaction mixture for a nonluciferase enzyme-mediatedreaction, and a derivative of luciferin which is a substrate for thenonluciferase enzyme, so as to yield a first mixture comprising aluminogenic product that is a substrate for a luciferase. In oneembodiment, the derivative is a compound of formula I. In anotherembodiment, the derivative is a compound of formula II. At least aportion of the first mixture is contacted with a second reaction mixturefor a luciferase-mediated reaction, so as to yield a second mixture.Then luminescence in the second reaction is detected or determined,thereby detecting or determining the presence or amount of a moleculefor the nonluciferase enzyme-mediated reaction in the sample, e.g.,compared to a control. In one embodiment, the derivative is a substratefor a transferase, e.g., glutathione S transferase (GST). Exemplaryderivatives useful to detect GST are shown in FIG. 12. In someembodiments, a second nonluciferase enzyme may be used to furtherchemically transform the product of the first nonluciferaseenzyme-mediated reaction to yield a substrate for a luciferase. Forexample, the sample, nonluciferase reaction mixture or luciferasereaction mixture may include an esterase, or the esterase may be addedseparately.

In one embodiment, derivatives of luciferin having an ester modificationare employed in methods of the invention, such as those to detectnonluciferase enzymes including P450 enzymes or monoamine oxidases(MAOs), or other enzymes such as flavin monoamine oxidases (FMOs),glutathione S transferases (GSTs), phosphatases, e.g., alkalinephosphatases (AP), or sulfatases, as those derivatives may have improvedproperties as a nonluciferase enzyme substrate. In particular, as shownherein, the addition of an ester group at the carboxy position of thethiazole ring of a luciferin derivative provided a substrate that wasrecognized by two P450 enzymes, 2D6 and 2C19, that did not react withpreviously described luciferin derivatives that are substrates ofcytochrome P450 enzymes (see U.S. published application 20040171099).

In an alternate embodiment, a method to detect or determine the presenceor amount of a molecule for a first nonluciferase enzyme-mediatedreaction in a sample is provided wherein the method includes contactinga sample, a reaction mixture for a nonluciferase-mediated enzymereaction and a luciferase-mediated reaction, and a derivative ofluciferin which is a substrate for the nonluciferase enzyme, yielding amixture. A reaction between the nonluciferase enzyme and the derivativeyields a luminogenic product that is a substrate for the luciferase.Luminescence in the mixture is detected or determined, thereby detectingor determining the presence or amount of a molecule for thenonluciferase-mediated reaction in the sample.

The invention also provides an embodiment directed to a method to detectthe presence or amount of a non-enzymatic molecule in a sample. Themethod includes contacting a sample, a first reaction mixture for anonenzyme-mediated reaction and a derivative of luciferin which in thepresence of the molecule yields a luminogenic product that is asubstrate for a luciferase, and contacting a portion of the firstreaction and a second reaction mixture for a luciferase-mediatedreaction, to yield a second reaction, then luminescence in the secondreaction is detected or determined, thereby detecting or determining thepresence or amount of the molecule. For instance, a mixture is providedhaving a sample, a first reaction mixture for a nonenzyme-mediatedreaction and a derivative of luciferin which in the presence of themolecule yields a luminogenic product that is a substrate for a beetleluciferase. At least a portion of the first mixture and a secondreaction mixture for a beetle luciferase-mediated reaction are mixed, toyield a second mixture, and then luminescence in the second mixture isdetected or determined, thereby detecting or determining the presence oramount of the molecule.

For the biolumingenic assays described herein which employ luciferinderivatives with a lower background luminescence than D-luciferin oraminoluciferin, those assays can use lower amounts, e.g., because smallchanges in luminescence can be detected, or higher amounts, e.g., inreactions that are improved by increased amounts of substrate, of thederivative, and those derivatives may have improved reactivity, e.g.,with a nonluciferase enzyme. In addition, for any of the bioluminogenicassays described herein, other reagents may be added to reactionmixtures, including but not limited to those that inhibit or preventinactivation of luciferase, or otherwise extend or enhance signal.

Also provided is a method to identify a modulator of a nonluciferaseenzyme-mediated reaction. The method includes contacting one or moreagents, a first reaction mixture for a nonluciferase enzyme-mediatedreaction, and a derivative of luciferin which is a substrate for thenonluciferase enzyme, so as to yield a first mixture, or providing sucha mixture. The first mixture in the absence of the one or more agentsincludes a luminogenic product that is a substrate for a beetleluciferase. At least a portion of the first mixture and a secondreaction mixture for a beetle luciferase-mediated reaction are mixed, soas to yield a second mixture. Luminescence in the second mixture iscompared with a control mixture, thereby identifying whether one or moreof the agents modulates the nonluciferase enzyme-mediated reaction.

In a further embodiment of the present invention, luciferin derivativesthat retain activity directly as a substrate for luciferase may beutilized to inactivate or inhibit the luciferase enzyme, e.g., bycovalent modification thereof, or as a competitive or noncompetitiveinhibitor thereof. This embodiment provides a method to screen forcompounds that prevent the inactivation of luciferase. Also provided isa method to identify a modulator of a luciferase-mediated reaction usinga luciferin derivative of the present invention.

The invention further provides a fluorogenic method which employs aderivative of a fluorophore that is modified to detect one or moremolecules, e.g., an enzyme, a cofactor for an enzymatic reaction such asATP, an enzyme substrate, an enzyme inhibitor, an enzyme activator, orOH radicals, or one or more conditions, e.g., redox conditions. Theinvention thus provides for bioluminogenic and fluorogenic assays todetect the amount, activity or presence of a molecule in a sample.

The invention also provides further embodiments directed to fluorogenicassays and derivatives of fluorophores which are a substrate for anonluciferase nonproteolytic enzyme. Thus, a method to detect ordetermine the presence or amount of a molecule for a nonproteaseenzyme-mediated reaction in a sample by a fluorescent method isprovided. The method includes contacting a sample, a first reactionmixture for a nonluciferase, nonproteolytic enzyme-mediated reaction,and a derivative of a fluorophore which includes a substrate for theenzyme, so as to yield a first mixture, wherein if the molecule ispresent in the sample, the first mixture comprises a hydroxy fluorescentproduct. The interaction between the enzyme and the derivativeoptionally produces an iminium and/or aldehyde intermediate whichoptionally undergoes a noncatalytic β-elimination to yield the hydroxyfluorescent product. Fluorescence in the mixture is detected ordetermined, thereby detecting or determining the presence or amount of amolecule for the nonprotease enzyme mediated reaction in the sample. Theinvention also provides for methods of using fluorophore derivatives ofthe invention to identify inhibitor, activators, substrates orco-factors for enzymatic reactions.

The invention provides compositions or kits having one or more luciferinderivatives and/or fluorophore derivatives of the invention. The kitsmay optionally contain other reagents, e.g., enzyme, reaction mixtures,and the like. The bioluminogenic reaction mixtures, compositions andkits of the invention may optionally include an agent that slows thereaction rate, e.g., amino methyl benzothiazole (AMBT) or aminophenylmethyl benzothiazol (APMBT), see U.S. published application 20040171099,yielding glow kinetics and/or an agent that stabilizes light production,e.g., a thiol or coenzyme A.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Numbering of ring atoms in the six membered ring (“A ring” or“ring A”), five membered center ring (“B ring” or “ring B”), and otherfive membered ring (“C ring” or “ring C”) of beetle luciferin(D-luciferin).

FIG. 2. Exemplary luciferin derivatives for enzymes.

FIG. 3. Structure of 5′-fluoroluciferin.

FIGS. 4A-B. Derivatives of luciferin useful as a redox sensor.

FIGS. 5A-B. Exemplary substrates.

FIG. 6. Redox substrates.

FIGS. 7A-B. Other exemplary derivatives of luciferin.

FIG. 8A. A graphical representation of RLU for reactions containing anesterase and a luciferin derivative, luciferin ethyl ester. 20 minuteincubation at 37° C.

FIG. 8B. A graphical representation of RLU for reactions containing anaminoluciferin derivative (isopropyl urethane aminoluciferin methylester; 50 μM), with and without esterase. 20 minute incubation at roomtemperature.

FIG. 9. A graphical representation of RLU for reactions containing anesterase and a luciferin derivative, luciferin methyl ester. 20 minuteincubation at 37° C.

FIG. 10. A graphical representation of RLU for reactions containing anesterase and a luciferin derivative, D-luciferin picolinyl ester. 20minute incubation at 37° C.

FIG. 11. A graphical representation of derivatives of fluorogenicderivatives useful as monoamine oxide (MAO) substrates.

FIGS. 12A-B. Luciferin derivatives useful to detect glutathione Stransferase (GST).

FIGS. 13A-D. A graphical representation of derivatives of luciferinderivatives for MAO assays. The derivative in FIG. 13B was not utilizedby MAO.

FIG. 14. Derivatives of luciferin useful as flavin monooxygenase (FMO)substrates.

FIG. 15. Exemplary FMO substrates.

FIG. 16. Exemplary AP substrates.

FIG. 17. RLU for a series of luciferin derivatives and each of a panelof P450 enzymes.

FIG. 18. RLU for a series of luciferin derivatives and each of a panelof P450 enzymes.

FIGS. 19A-C. Structures of exemplary derivatives useful to detect P450enzymes.

FIG. 20. A graphical representation of CYP2D6 and CYP2C19 activityagainst luciferin-ME. A) RLU in reactions with luciferin-ME. B) Signalto background ratio in reactions with luciferin-ME.

FIG. 21. A graphical representation of P450 activities with luciferin-MEmethyl ester.

FIG. 22. A graphical representation of CYP2D6 activity with luciferin-MEpicolinyl ester with and without esterase. Reactions contained 1 pmolCYP2D6.

FIG. 23. A graphical representation of CYP2C19 activity withluciferin-H-picolinylester with and without esterase. Reactionscontained 1 pmol CYP2C19.

FIG. 24. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative, D-luciferin ethylester with a C ring modification. Reactions contained 1 pmol P450.

FIG. 25. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative, luciferin methylester with a C ring modification. Reactions contained 1 pmol P450 or 5pmol CYP19.

FIG. 26. Graphical representation of RLU for reactions containing one ofthree P450 enzymes and luciferin-ME-ethylene glycol ester, with orwithout esterase.

FIG. 27A. A graphical representation of RLU for an aminoluciferinderivative (N-isopropylaminoluciferin) (50 μM) in a luciferase reaction.

FIG. 27B. A graphical representation of RLU for an aminoluciferinderivative (dimethyl aminoluciferin; 100 μM) and aminoluciferin (100 μM)in a luciferase reaction.

FIG. 28. A graphical representation of signal to background ratio for areaction between 4′,6′ dimethyl ether luciferin and each of a panel ofP450 enzymes. Reactions contained 1 pmol P450.

FIG. 29. A graphical representation of signal background ratio for areaction between a luciferin derivative,(2-(6-methoxyquinolin-2-yl)-4,5-dihydrothiazole-4-carboxylate) and eachof a panel of P450 enzymes. Reactions contained 1 pmol P450.

FIG. 30. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,2-(7-methyoxy-quinoxalin-2-yl)-4-5-dihydro-thiazole-4-carboxylic acid.Reactions contained 1 pmol P450.

FIG. 31. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,(N-((3-chloro-4-(2-(dimethylamino)-1-phenylpropoxy)benzyloxy)carbonyl)-6′-aminoluciferin).Reactions contained 1 pmol P450 or 5 pmol CYP19.

FIG. 32. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative, luciferin4-piperidinemethyl ether. Reactions contained 1 pmol P450.

FIG. 33. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,N-(2-chloroethoxycarbonyl)-aminoluciferin. Reactions contained 1 pmolP450.

FIG. 34. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,4′-(N,N-dimethylamino)methyl-6′(O-methyl) luciferin. Reactions contained1 pmol P450.

FIG. 35. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,4-(N,N-dimethylaminomethyl)-6-methoxyquinolyl luciferin. Reactionscontained 1 pmol P450.

FIG. 36. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative, luciferin2-(1-piperazine)ethyl ether. Reactions contained 1 pmol P450.

FIG. 37A. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative, meta-N-methylpicolinyl luciferin methyl ester. Reactions contained 1 pmol P450. A 30minute incubation at 37° C. was followed by a 20 minute incubation atroom temperature with or without esterase in luciferin detectionreagent.

FIG. 37B. A graphical representation of RLU for reactions containing aluciferin derivative, meta-N-methyl picolinyl luciferin methyl ester,with or without esterase. 20 minute incubation at room temperature.

FIG. 38. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,methamphetamine-luciferin. Reactions contained 1 pmol P450.

FIG. 39. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,N-isobutoxycarbonyl-aminoluciferin. Reactions contained 1 pmol P450.

FIG. 40. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative, luciferin-Hmethyl ester and esterase. Reactions contained 1 pmol P450.

FIG. 41. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative, luciferin-MEethylene glycolester. Reactions contained 1 pmol P450, 5 pmol CYP19, or40 μg HLM.

FIG. 42. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative, methoxyquinolylluciferin methyl ester. Reactions contained 1 pmol P450.

FIG. 43. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,6′(o-trifluoromethylbenzyloxy)-luciferin. 1 pmol P450 or 5 pmol CYP19.

FIG. 44. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,6′methoxy-luciferin-hydrazide. Reactions contain 1 pmol of P450 or 5pmol CYP19.

FIG. 45. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,6′-(3-((4-phenylpiperizin-1-yl))methyl)benzyloxy)-luciferin. Reactionscontained 1 pmol P450 or 20 μg HLM.

FIG. 46. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,6′-(2,4,6-trimethylbenzyloxy)-luciferin. 1 pmol of P450 or 5 pmol CYP19.

FIG. 47. A graphical representation of RLU for reactions containing oneof a panel of P450 enzymes and a luciferin derivative,6′(4-chlorophenylthio)methoxyluciferin. Reactions contained 1 pmol ofP450 or 5 pmol CYP19.

FIG. 48. Inhibition of CYP3A4 activity by troleanomycin in a reactionwith luciferin-F₅BE (6′-(2,3,4,5,6 pentafluorobenzyloxy)-luciferin).

FIG. 49. Inhibition of CYP3A4 activity by nifedipine in a reaction withluciferin-PPX4FE(6′-(2,3,4,6-tetrafluoro-5-((4-phenylpiperazin-1-yl)methyl)benzyloxy)-luciferin).

FIG. 50. Inhibition of CYP3A4 activity by nifedipine in a reaction withluciferin-PPXE(6′-(3-((4-phenylpiperazin-1-yl)methyl)benzyloxy)-luciferin.

FIG. 51. CYP3A4 activity in human hepatocytes hepatocytes aftertreatment with rifampicin, ketoconazole, or rifampicin and ketoconazole,in a reaction with luciferin-PPXE.

FIG. 52. CYP3A4 activity in human hepatocytes after treatment withrifampicin, ketoconazole, or rifampicin and ketoconazole, in a reactionwith luciferin-F₅BE.

FIG. 53. Luciferin derivatives which may be luciferase substrates:N-isopropyl aminoluciferin bisethyl aminoluciferin; andbenzylaminoluciferin.

FIG. 54. Structures of derivatives of luciferin which may be a substratefor different luciferases.

FIG. 55. A graphical representation of RLU for luciferin or5-fluoroluciferin in luciferase reactions at different pHs.

FIG. 56. ATP titration of a 5′ fluoroluciferin and luciferin.

FIG. 57. ATP titration of a 7′ fluoroluciferin.

FIG. 58. Spectra of various fluoroluciferins.

FIG. 59. pH titration of a 7′ fluoroluciferin.

FIG. 60. Luciferin derivatives useful as luciferase substrates andscaffolds for luciferin derivatives useful as nonluciferase substrates.

DETAILED DESCRIPTION OF THE INVENTION I. Definitions

As used herein, the following terms and expressions have the indicatedmeanings. It will be appreciated that the compounds of the presentinvention contain asymmetrically substituted carbon atoms, and may beisolated in optically active or racemic forms. It is well known in theart how to prepare optically active forms, such as by resolution ofracemic forms or by synthesis from optically active starting materials.All chiral, diastereomeric, racemic forms and all geometric isomericforms of a structure are part of this invention.

Specific values listed below for radicals, substituents, and ranges, arefor illustration only; they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

As used herein, the term “substituted” is intended to indicate that oneor more (e.g., 1, 2, 3, 4, or 5; in some embodiments 1, 2, or 3; and inother embodiments 1 or 2) hydrogens on the group indicated in theexpression using “substituted” is replaced with a selection from theindicated group(s), or with a suitable group known to those of skill inthe art, provided that the indicated atom's normal valency is notexceeded, and that the substitution results in a stable compound.Suitable indicated groups include, e.g., alkyl, alkenyl, alkynyl,alkoxy, halo, haloalkyl, hydroxy, hydroxyalkyl, aryl, heteroaryl,heterocycle, cycloalkyl, alkanoyl, alkoxycarbonyl, amino, alkylamino,dialkylamino, trifluoromethylthio, difluoromethyl, acylamino, nitro,trifluoromethyl, trifluoromethoxy, carboxy, carboxyalkyl, keto, thioxo,alkylthio, alkylsulfinyl, alkylsulfonyl, arylsulfinyl, arylsulfonyl,heteroarylsulfinyl, heteroarylsulfonyl, heterocyclesulfinyl,heterocyclesulfonyl, phosphate, sulfate, hydroxylamine, hydroxyl(alkyl)amine, and cyano. Additionally, the suitable indicated groups caninclude, e.g., —X, —R, —O⁻, —OR, —SR, —S⁻, —NR₂, —NR₃, ═NR, —CX₃, —CN,—OCN, —SCN, —N═C═O, —NCS, —NO, —NO₂, ═N₂, —N₃, NC(═O)R, —C(═O)R,—C(═O)NRR —S(═O)₂O⁻, —S(═O)₂OH, —S(═O)₂R, —OS(═O)₂OR, —S(═O)₂NR,—S(═O)R, —OP(═O)O₂RR, —P(═O)O₂RR —P(═O)(O⁻)₂, —P(═O)(OH)₂, —C(═O)R,—C(═O)X, —C(S)R, —C(O)OR, —C(O)O⁻, —C(S)OR, —C(O)SR, —C(S)SR, —C(O)NRR,—C(S)NRR, —C(NR)NRR, where each X is independently a halogen (“halo”):F, Cl, Br, or I; and each R is independently H, alkyl, aryl, heteroaryl,heterocycle, a protecting group or prodrug moiety. As would be readilyunderstood by one skilled in the art, when a substituent is keto (═O) orthioxo (═S), or the like, then two hydrogen atoms on the substitutedatom are replaced.

“Stable compound” and “stable structure” are meant to indicate acompound that is sufficiently robust to survive isolation to a usefuldegree of purity from a reaction mixture. Only stable compounds arecontemplated by and claimed in the present invention, however, certainunstable compounds, for example, those that cannot easily be isolated,can be employed in the methods described herein.

One diastereomer may display superior properties or activity comparedwith another. When required, separation of the racemic material can beachieved by HPLC using a chiral column or by a resolution using aresolving agent such as camphonic chloride as described by Thomas J.Tucker, et al., J. Med. Chem. 1994, 37, 2437-2444. A chiral compound mayalso be directly synthesized using a chiral catalyst or a chiral ligand,e.g. Mark A. Huffman, et al., J. Org. Chem. 1995, 60, 1590-1594.

As used herein, the term “alkyl” refers to a branched, unbranched, orcyclic hydrocarbon having, for example, from 1 to 30 carbon atoms, andoften 1 to 12, or 1 to about 6 carbon atoms. Examples include, but arenot limited to, methyl, ethyl, 1-propyl, 2-propyl, 1-butyl,2-methyl-1-propyl, 2-butyl, 2-methyl-2-propyl(t-butyl), 1-pentyl,2-pentyl, 3-pentyl, 2-methyl-2-butyl, 3-methyl-2-butyl,3-methyl-1-butyl, 2-methyl-1-butyl, 1-hexyl, 2-hexyl, 3-hexyl,2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl,3-methyl-3-pentyl, 2-methyl-3-pentyl, 2,3-dimethyl-2-butyl,3,3-dimethyl-2-butyl, hexyl, octyl, decyl, dodecyl, and the like. Thealkyl can be unsubstituted or substituted. The alkyl can also beoptionally partially or fully unsaturated. As such, the recitation of analkyl group includes both alkenyl and alkynyl groups. The alkyl can be amonovalent hydrocarbon radical, as described and exemplified above, orit can be a divalent hydrocarbon radical (i.e., alkylene).

The term “alkenyl” refers to a monoradical branched or unbranchedpartially unsaturated hydrocarbon chain (i.e. a carbon-carbon, sp²double bond). In one embodiment, an alkenyl group can have from 2 to 10carbon atoms, or 2 to 6 carbon atoms. In another embodiment, the alkenylgroup has from 2 to 4 carbon atoms. Examples include, but are notlimited to, ethylene or vinyl, allyl, cyclopentenyl, 5-hexenyl, and thelike. The alkenyl can be unsubstituted or substituted.

The term “alkynyl” refers to a monoradical branched or unbranchedhydrocarbon chain, having a point of complete unsaturation (i.e. acarbon-carbon, sp triple bond). In one embodiment, the alkynyl group canhave from 2 to 10 carbon atoms, or 2 to 6 carbon atoms. In anotherembodiment, the alkynyl group can have from 2 to 4 carbon atoms. Thisterm is exemplified by groups such as ethynyl, 1-propynyl, 2-propynyl,1-butynyl, 2-butynyl, 3-butynyl, 1-hexynyl, 2-hexynyl, 3-hexynyl,1-octynyl, and the like. The alkynyl can be unsubstituted orsubstituted.

The term “cycloalkyl” refers to cyclic alkyl groups of from 3 to 10carbon atoms having a single cyclic ring or multiple condensed rings.Such cycloalkyl groups include, by way of example, single ringstructures such as cyclopropyl, cyclobutyl, cyclopentyl, cyclooctyl, andthe like, or multiple ring structures such as adamantanyl, and the like.The cycloalkyl can be unsubstituted or substituted. The cycloalkyl groupcan be monovalent or divalent, and can be optionally substituted asdescribed above for alkyl groups. The cycloalkyl group can optionallyinclude one or more cites of unsaturation, for example, the cycloalkylgroup can include one or more carbon-carbon double bonds, such as, forexample, cyclohexene, 1,3-cyclohexadiene, 1,4-cyclohexadiene, and thelike.

The term “alkoxy” refers to the group alkyl-O—, where alkyl is asdefined herein. In one embodiment, alkoxy groups include, e.g., methoxy,ethoxy, n-propoxy, iso-propoxy, n-butoxy, tert-butoxy, sec-butoxy,n-pentoxy, n-hexoxy, 1,2-dimethylbutoxy, and the like. The alkoxy can beunsubstituted or substituted.

As used herein, “aryl” refers to an aromatic hydrocarbon group derivedfrom the removal of one hydrogen atom from a single carbon atom of aparent aromatic ring system. The radical can be at a saturated orunsaturated carbon atom of the parent ring system. The aryl group canhave from 6 to 30 carbon atoms. The aryl group can have a single ring(e.g., phenyl) or multiple condensed (fused) rings, wherein at least onering is aromatic (e.g., naphthyl, dihydrophenanthrenyl, fluorenyl, oranthryl). Typical aryl groups include, but are not limited to, radicalsderived from benzene, naphthalene, anthracene, biphenyl, and the like.The aryl can be unsubstituted or optionally substituted, as describedabove for alkyl groups.

The term “halo” refers to fluoro, chloro, bromo, and iodo. Similarly,the term “halogen” refers to fluorine, chlorine, bromine, and iodine.

The term “haloalkyl” refers to alkyl as defined herein substituted by 1or more halo groups as defined herein, which may be the same ordifferent. In one embodiment, the haloalkyl can be substituted with 1,2, 3, 4, or 5 halo groups. In another embodiment, the haloalkyl can bysubstituted with 1, 2, or 3 halo groups. The term haloalkyl also includeperfluoro-alkyl groups. Representative haloalkyl groups include, by wayof example, trifluoromethyl, 3-fluorododecyl, 12,12,12-trifluorododecyl,2-bromooctyl, 3-bromo-6-chloroheptyl, 1H,1H-perfluorooctyl, and thelike. The haloalkyl can be optionally substituted as described above foralkyl groups.

The term “heteroaryl” is defined herein as a monocyclic, bicyclic, ortricyclic ring system containing one, two, or three aromatic rings andcontaining at least one nitrogen, oxygen, or sulfur atom in an aromaticring, and that can be unsubstituted or substituted, for example, withone or more, and in particular one to three, substituents, as describedabove in the definition of “substituted”. Typical heteroaryl groupscontain 2-20 carbon atoms in addition to the one or more hetoeroatoms.Examples of heteroaryl groups include, but are not limited to,2H-pyrrolyl, 3H-indolyl, 4H-quinolizinyl, acridinyl, benzo[b]thienyl,benzothiazolyl, β-carbolinyl, carbazolyl, chromenyl, cinnolinyl,dibenzo[b,d]furanyl, furazanyl, furyl, imidazolyl, imidizolyl,indazolyl, indolisinyl, indolyl, isobenzofuranyl, isoindolyl,isoquinolyl, isothiazolyl, isoxazolyl, naphthyridinyl, oxazolyl,perimidinyl, phenanthridinyl, phenanthrolinyl, phenarsazinyl,phenazinyl, phenothiazinyl, phenoxathiinyl, phenoxazinyl, phthalazinyl,pteridinyl, purinyl, pyranyl, pyrazinyl, pyrazolyl, pyridazinyl,pyridyl, pyrimidinyl, pyrimidinyl, pyrrolyl, quinazolinyl, quinolyl,quinoxalinyl, thiadiazolyl, thianthrenyl, thiazolyl, thienyl, triazolyl,tetrazolyl, and xanthenyl. In one embodiment the term “heteroaryl”denotes a monocyclic aromatic ring containing five or six ring atomscontaining carbon and 1, 2, 3, or 4 heteroatoms independently selectedfrom non-peroxide oxygen, sulfur, and N(Z) wherein Z is absent or is H,O, alkyl, aryl, or (C₁-C₆)alkylaryl. In another embodiment heteroaryldenotes an ortho-fused bicyclic heterocycle of about eight to ten ringatoms derived therefrom, particularly a benz-derivative or one derivedby fusing a propylene, trimethylene, or tetramethylene diradicalthereto.

The term “heterocycle” refers to a saturated or partially unsaturatedring system, containing at least one heteroatom selected from the groupoxygen, nitrogen, and sulfur, and optionally substituted with one ormore groups as defined herein under the term “substituted”. Aheterocycle can be a monocyclic, bicyclic, or tricyclic group containingone or more heteroatoms. A heterocycle group also can contain an oxogroup (═O) or a thioxo (═S) group attached to the ring. Non-limitingexamples of heterocycle groups include 1,3-dihydrobenzofuran,1,3-dioxolane, 1,4-dioxane, 1,4-dithiane, 2H-pyran, 2-pyrazoline,4H-pyran, chromanyl, imidazolidinyl, imidazolinyl, indolinyl,isochromanyl, isoindolinyl, morpholine, piperazinyl, piperidine,piperidyl, pyrazolidine, pyrazolidinyl, pyrazolinyl, pyrrolidine,pyrroline, quinuclidine, and thiomorpholine.

The term “heterocycle” can include, by way of example and notlimitation, a monoradical of the heterocycles described in Paquette, LeoA.; Principles of Modern Heterocyclic Chemistry (W. A. Benjamin, NewYork, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistryof Heterocyclic Compounds, A Series of Monographs” (John Wiley & Sons,New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and28; and J. Am. Chem. Soc. 1960, 82, 5566. In one embodiment,“heterocycle” includes a “carbocycle” as defined herein, wherein one ormore (e.g. 1, 2, 3, or 4) carbon atoms have been replaced with aheteroatom (e.g. O, N, or S).

Examples of heterocycles, by way of example and not limitation, include,dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, piperidinyl, 4-piperidonyl,pyrrolidinyl, 2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl,tetrahydroquinolinyl, tetrahydroisoquinolinyl, decahydroquinolinyl,octahydroisoquinolinyl, azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl,2H,6H-1,5,2-dithiazinyl, thienyl, thianthrenyl, pyranyl,isobenzofuranyl, chromenyl, xanthenyl, phenoxathinyl, 2H-pyrrolyl,isothiazolyl, isoxazolyl, pyrazinyl, pyridazinyl, indolizinyl,isoindolyl, 3H-indolyl, 1H-indazoly, purinyl, 4H-quinolizinyl,phthalazinyl, naphthyridinyl, quinoxalinyl, quinazolinyl, cinnolinyl,pteridinyl, carbazolyl, β-carbolinyl, phenanthridinyl, acridinyl,pyrimidinyl, phenanthrolinyl, phenazinyl, phenothiazinyl, furazanyl,phenoxazinyl, isochromanyl, chromanyl, imidazolidinyl, imidazolinyl,pyrazolidinyl, pyrazolinyl, piperazinyl, indolinyl, isoindolinyl,quinuclidinyl, morpholinyl, oxazolidinyl, benzotriazolyl,benzisoxazolyl, oxindolyl, benzoxazolinyl, isatinoyl, andbis-tetrahydrofuranyl.

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Carbon bonded heterocycles include 2-pyridyl, 3-pyridyl,4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl, 4-pyridazinyl,5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl, 4-pyrimidinyl,5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl, 5-pyrazinyl,6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, and the like.

By way of example and not limitation, nitrogen bonded heterocycles canbe bonded at position 1 of an aziridine, azetidine, pyrrole,pyrrolidine, 2-pyrroline, 3-pyrroline, imidazole, imidazolidine,2-imidazoline, 3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline,3-pyrazoline, piperidine, piperazine, indole, indoline, 1H-indazole,position 2 of a isoindole, or isoindoline, position 4 of a morpholine,and position 9 of a carbazole, or β-carboline. In one embodiment,nitrogen bonded heterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl,1-imidazolyl, 1-pyrazolyl, and 1-piperidinyl.

The term “carbocycle” refers to a saturated, unsaturated or aromaticring having 3 to 8 carbon atoms as a monocycle, 7 to 12 carbon atoms asa bicycle, and up to about 30 carbon atoms as a polycycle. Monocycliccarbocycles typically have 3 to 6 ring atoms, still more typically 5 or6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g.,arranged as a bicyclo [4,5], [5,5], [5,6] or [6,6] system, or 9 or 10ring atoms arranged as a bicyclo [5,6] or [6,6] system. Examples ofcarbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, phenyl, spiryland naphthyl. The carbocycle can be optionally substituted as describedabove for alkyl groups.

The term “alkanoyl” or “alkylcarbonyl” refers to —C(═O)R, wherein R isan alkyl group as previously defined.

The term “acyloxy” or “alkylcarboxy” refers to —O—C(═O)R, wherein R isan alkyl group as previously defined. Examples of acyloxy groupsinclude, but are not limited to, acetoxy, propanoyloxy, butanoyloxy, andpentanoyloxy. Any alkyl group as defined above can be used to form anacyloxy group.

The term “alkoxycarbonyl” refers to —C(═O)OR (or “COOR”), wherein R isan alkyl group as previously defined.

The term “amino” refers to —NH₂. The amino group can be optionallysubstituted as defined herein for the term “substituted”. The term“alkylamino” refers to —NR₂, wherein at least one R is alkyl and thesecond R is alkyl or hydrogen. The term “acylamino” refers toN(R)C(═O)R, wherein each R is independently hydrogen, alkyl, or aryl.

The term “amino acid,” includes a residue of a natural amino acid (e.g.Ala, Arg, Asn, Asp, Cys, Glu, Gln, Gly, His, Hyl, Hyp, Ile, Leu, Lys,Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val) in D or L form, as well asunnatural amino acids (e.g. phosphoserine, phosphothreonine,phosphotyrosine, hydroxyproline, gamma-carboxyglutamate; hippuric acid,octahydroindole-2-carboxylic acid, statine,1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, penicillamine,ornithine, citruline, α-methyl-alanine, para-benzoylphenylalanine,phenylglycine, propargylglycine, sarcosine, and tert-butylglycine). Theterm also comprises natural and unnatural amino acids bearing aconventional amino protecting group (e.g. acetyl or benzyloxycarbonyl),as well as natural and unnatural amino acids protected at the carboxyterminus (e.g. as a (C₁-C₆)alkyl, phenyl or benzyl ester or amide; or asan α-methylbenzyl amide). Other suitable amino and carboxy protectinggroups are known to those skilled in the art (See for example, Greene,T. W.; Wutz, P. G. M. Protecting Groups In Organic Synthesis, 2^(nd)edition, John Wiley & Sons, Inc., New York (1991) and references citedtherein).

The term “peptide” describes a sequence of 2 to 35 amino acids (e.g. asdefined hereinabove) or peptidyl residues. The sequence may be linear orcyclic. For example, a cyclic peptide can be prepared or may result fromthe formation of disulfide bridges between two cysteine residues in asequence. Preferably a peptide comprises 3 to 20, or 5 to 15 aminoacids. Peptide derivatives can be prepared as disclosed in U.S. Pat.Nos. 4,612,302; 4,853,371; and 4,684,620, or as described in theExamples herein below. Peptide sequences specifically recited herein arewritten with the amino terminus on the left and the carboxy terminus onthe right.

The term “saccharide” refers to a sugar or other carbohydrate,especially a simple sugar. The saccharide can be a C₆-polyhydroxycompound, typically C₆-pentahydroxy, and often a cyclic glycal. The termincludes the known simple sugars and their derivatives, as well aspolysaccharides with two or more monosaccaride residues. The saccharidecan include protecting groups on the hydroxyl groups, as described abovein the definition of amino acids. The hydroxyl groups of the saccharidecan be replaced with one or more halo or amino groups. Additionally, oneor more of the carbon atoms can be oxidized, for example to keto orcarboxyl groups.

The term “interrupted” indicates that another group is inserted betweentwo adjacent carbon atoms (and the hydrogen atoms to which they areattached (e.g., methyl (CH₃), methylene (CH₂) or methine (CH))) of aparticular carbon chain being referred to in the expression using theterm “interrupted, provided that each of the indicated atoms' normalvalency is not exceeded, and that the interruption results in a stablecompound. Suitable groups that can interrupt a carbon chain include,e.g., with one or more non-peroxide oxy (—O—), thio (—S—), imino(—N(H)—), methylene dioxy (—OCH₂O—), carbonyl (—C(═O)—), carboxy(—C(═O)O—), carbonyldioxy (—OC(═O)O—), carboxylato (—OC(═O)—), imine(C═NH), sulfinyl (SO) and sulfonyl (SO₂). Alkyl groups can beinterrupted by one or more (e.g., 1, 2, 3, 4, 5, or about 6) of theaforementioned suitable groups. The site of interruption can also bebetween a carbon atom of an alkyl group and a carbon atom to which thealkyl group is attached.

As to any of the above groups, which contain one or more substituents,it is understood, of course, that such groups do not contain anysubstitution or substitution patterns that are sterically impracticaland/or synthetically non-feasible. In addition, the compounds of thisinvention include all stereochemical isomers arising from thesubstitution of these compounds.

Selected substituents within the compounds described herein are presentto a recursive degree. In this context, “recursive substituent” meansthat a substituent may recite another instance of itself. Because of therecursive nature of such substituents, theoretically, a large number maybe present in any given claim. One of ordinary skill in the art ofmedicinal chemistry and organic chemistry understands that the totalnumber of such substituents is reasonably limited by the desiredproperties of the compound intended. Such properties include, by ofexample and not limitation, physical properties such as molecularweight, solubility or log P, application properties such as activityagainst the intended target, and practical properties such as ease ofsynthesis.

Recursive substituents are an intended aspect of the invention. One ofordinary skill in the art of medicinal and organic chemistry understandsthe versatility of such substituents. To the degree that recursivesubstituents are present in an claim of the invention, the total numberwill be determined as set forth above.

The term “linker” as used herein is a carbon chain that covalentlyattaches two chemical groups together and optionally can self-cleave orif covalently bonded to a substrate for an enzyme, may be cleaved bythat enzyme or another molecule, which chain is optionally interruptedby one or more nitrogen atoms, oxygen atoms, carbonyl groups,(substituted)aromatic rings, or peptide bonds.

The term “luciferase,” unless specified otherwise, refers to a naturallyoccurring or mutant luciferase. The luciferase, if naturally occurring,may be obtained easily by the skilled from an organism. If theluciferase is one that occurs naturally or is a mutant, which retainsactivity in the luciferase-luciferin reaction, of a naturally occurringluciferase, it can be obtained readily from a culture of bacteria,yeast, mammalian cells, insect cells, plant cells, or the like,transformed to express a cDNA encoding the luciferase, or from an invitro cell-free system for making the luciferase from a nucleic acidencoding same. Luciferases are available from Promega Corporation,Madison, Wis.

As used herein, a “fluorophore” includes a molecule which is capable ofabsorbing energy at a wavelength range and releasing energy at awavelength range other than the absorbance range. The term “excitationwavelength” refers to the range of wavelengths at which a fluorophoreabsorbs energy. The term “emission wavelength” refers to the range ofwavelengths that the fluorophore releases energy or fluoresces.

As used herein, a “bioluminogenic assay” or “bioluminogenic reaction”includes a reaction in which a product of a reaction between anonluciferase enzyme and a derivative of luciferin or aminoluciferin isa substrate for luciferase or a product of a nonenzymatic reactionhaving a derivative of luciferin or aminoluciferin is a substrate forluciferase, or a reaction between a luciferase and a derivative ofluciferin or aminoluciferin, is bioluminogenic, i.e., produces ameasurable amount of light.

As used herein, “bioluminescence” is light produced as a result of areaction between an enzyme and a substrate that generates light.Examples of such enzymes (bioluminescent enzymes) include fireflyluciferase, click beetle luciferase, Renilla luciferase, cypridinaluciferase, Aequorin photoprotein, obelin photoprotein and the like.

As used herein, a “bioluminogenic assay reagent” may include asubstrate, as well as a cofactor(s) or other molecule(s) such as aprotein, e.g., an enzyme, for a bioluminogenic reaction.

A “reaction mixture” may contain all reagents for a particular reaction,or may lack at least one of the reagents for the reaction. For example,a luciferase reaction mixture may contain reagents for the reactionexcept for a substrate for the luciferase, e.g., a reaction mixtureuseful to determine whether a test sample has a luciferase substrate. Areaction mixture for a nonluciferase enzyme may include all reagents forthat reaction except for a molecule to be detected, e.g., the mixturecontains all reagents except for a cofactor for the nonluciferaseenzyme, and so the mixture is useful to detect the presence of thecofactor in a test sample.

As used herein a “derivative of luciferin” or a “derivative ofaminoluciferin” is a molecule that is a substrate for a nonluciferaseenzyme and a prosubstrate of a luciferase, a substrate for a luciferase,a substrate for a nonluciferase enzyme and a substrate for a luciferase,or is useful to detect molecules generated in nonenzymatic reactions.The derivatives of the invention have one or more modifications to oneor more of the three rings and/or substituents attached to one or moreof the rings of the D-luciferin or aminoluciferin backbone (see FIG. 1).

A “fluorogenic assay” or “fluorogenic reaction” includes a reaction inwhich a product of a reaction between a nonluciferase, nonproteolyticenzyme and a derivative of a fluorophore is fluorescent. A “fluorogenicassay reagent” may include a substrate, as well as a cofactor(s) orother molecule(s) such as a protein, e.g., an enzyme, for a fluorogenicreaction. Thus, the invention provides a method to detect or determinethe presence or amount of a molecule for a nonprotease enzyme-mediatedreaction in a sample.

II. Methods of the Invention

The invention provides a bioluminogenic or fluorogenic method whichemploys a derivative of luciferin or aminoluciferin or a derivative of afluorophore to detect one or more molecules, e.g., an enzyme, a cofactorfor an enzymatic reaction such as ATP, an enzyme substrate, an enzymeinhibitor, an enzyme activator, or OH radicals, or one or moreconditions, e.g., redox conditions. The invention thus provides forbioluminogenic and fluorogenic assays to detect the amount, activity orpresence of a molecule in a sample.

The methods may be used, for example, to determine the presence oramount of at least one molecule, e.g., a nonluciferase enzyme, aregulator of a nonluciferase enzyme, a nonluciferase enzyme substrate,and/or cofactors of the reaction, or a condition in a sample includingbut not limited to an animal, e.g., vertebrate, physiological fluid,e.g., blood, plasma, urine, mucous secretions and the like, a cell, celllysate, cell supernatant, or purified fraction of a cell (e.g., asubcellular fraction). In one embodiment, the methods according to thepresent invention provide a rapid method for detecting one or moremolecules in a single sample such as an aliquot of cells or a lysatethereof. In one embodiment, the method includes quantifying thepresence, amount or specific activity of a molecule such as an enzyme,substrate or cofactor in a bioluminogenic assay or quantifying thepresence or amount of an enzyme, substrate or cofactor in a fluorogenicassay. The intensity of the bioluminogenic or fluorogenic signal is afunction of the presence or amount of the respective molecule. Inaddition, the reaction may contain one or more test agents, e.g., enzymeinhibitors or activators, and/or different concentrations of inhibitorsor activators. In one embodiment, the method employs at least twodifferent reactions, where the first reaction is a nonluciferaseenzyme-mediated reaction and the second reaction is a beetleluciferase-mediated reaction. In another embodiment, the first reactionis a nonenzymatic reaction and the second reaction is a beetleluciferase-mediated reaction. In yet another embodiment, the methodemploys a single reaction, e.g., a beetle luciferase-mediated reactionor a fluorogenic reaction.

Thus, a bioluminogenic assay may directly or indirectly detect, e.g.,measure, the amount, presence or specific activity of, for example, acofactor for an enzyme-mediated reaction, an enzyme, an enzymesubstrate, an inhibitor of the enzyme, an activator of the enzyme, or acondition. For instance, in one embodiment, a beetle luciferase and aderivative of luciferin that is a substrate of the beetle luciferase maybe employed in a bioluminogenic assay to detect ATP concentration. Inanother embodiment, a derivative of luciferin which is a substrate for anonluciferase enzyme, for instance, a derivative which is a substrate ofa monoamine oxidase, yields a product which is a substrate for a beetleluciferase, and so may be employed in a bioluminogenic assay to detectthe oxidase. In one embodiment, the derivative is a prosubstrate of abeetle luciferase, which yields a product that is a substrate ofluciferase but does not itself yield a substantial amount of light in areaction with the beetle luciferase. In some embodiments, the derivativeis a substrate for a nonluciferase enzyme or useful to detect anothermolecule, and a substrate for luciferase which yields a substantialamount of light. In this embodiment, the derivative is altered byluciferase but is generally inefficient in a light generating reaction.

In one embodiment, the invention provides a bioluminescent assay methodto detect one or more nonluciferase enzymes. The method includescontacting a sample suspected of having one or more nonluciferaseenzymes, or a substrate or a co-factor for the nonluciferase-mediatedreaction, with a corresponding reaction mixture that includes aderivative of luciferin or a derivative of aminoluciferin that is asubstrate for the nonluciferase enzyme. In one embodiment, thederivative is one having a modification in the A ring of D-luciferinthat includes a recognition site for the nonluciferase enzyme, e.g., fora phosphatase. In another embodiment, the derivative is one having amodification in the B ring of D-luciferin which derivative is asubstrate for luciferase or a prosubstrate for luciferase. In anotherembodiment, the derivative is one having a modification in the C ring ofluciferin that includes a recognition site for an enzyme of interest,e.g., acetylcholinesterase. In another embodiment, the derivative is onehaving a modification in one of the rings that includes a recognitionsite for the enzyme of interest, as well as a further modification inthat ring or one or more of the other rings.

For derivatives that are a substrate for luciferase, as well asoptionally a substrate of a nonluciferase enzyme or other molecules, thederivative may be employed to detect luciferase, or a co-factor,inhibitor, or activator of the luciferase reaction. If the derivative isa prosubstrate for luciferase, i.e., the product of a reaction betweenthe derivative and the nonluciferase enzyme is a substrate forluciferase, sequential or concurrent reactions for the nonluciferaseenzyme and the luciferase may be conducted. For instance, a reaction fora nonluciferase enzyme that contains the prosubstrate may be conductedin a single well and a beetle luciferase reaction mixture added to thatwell. In another embodiment, a reaction mixture for a nonluciferaseenzyme that contains the prosubstrate is conducted in a single well anda portion of that reaction added to a different well having a beetleluciferase reaction mixture. Alternatively, reactions may be conductedsimultaneously in the same well.

The invention thus provides a method to detect or determine the presenceor amount of a molecule for a nonluciferase enzyme-mediated reaction ina sample. The method includes contacting a sample, a first reactionmixture for a nonluciferase enzyme-mediated reaction, and a derivativeof luciferin which is a substrate for the nonluciferase enzyme, so as toyield a first mixture or providing such a first mixture comprising aluminogenic product that is a substrate for a luciferase, or providingsuch a first mixture. In one embodiment, the derivative is a compound offormula I. In another embodiment, the derivative is a compound offormula II. At least a portion of the first mixture is contacted with asecond reaction mixture for a beetle luciferase-mediated reaction, so asto yield a second mixture. Then luminescence in the second mixture isdetected or determined, thereby detecting or determining the presence oramount of a molecule for the nonluciferase enzyme-mediated reaction inthe sample. In some embodiments, the nonluciferase reaction mixture orluciferase reaction mixture may include an esterase, e.g., if theproduct of the reaction between the derivative and the nonluciferaseenzyme has an ester group and that product is a proluciferase substrate.The esterase may be included with the first reaction mixture, addedprior to initiation of the luciferase reaction mixture, or included inthe luciferase reaction mixture. In some embodiments, e.g., a derivativewith a picolinyl ester, the product of the reaction between thederivative and the nonluciferase enzyme is a substrate for luciferase inthe absence of an exogenously added esterase. In one embodiment,derivatives of luciferin having an ester modification are employed inmethods of the invention, such as those to detect nonluciferase enzymesincluding cytochrome P450 enzymes, as those derivatives may haveimproved properties as a nonluciferase substrate. Although not intendingto be bound by any mechanism, the inclusion of the ester modification atposition 5 in ring C may block negative charges or add a lipophilicquality to the derivative, rendering it an improved substrate.

Further provided is a method to detect or determine the presence oramount of a molecule for a nonluciferase enzyme-mediated reaction in asample. The method includes contacting a sample, a reaction mixture fora nonluciferase-mediated enzyme reaction and for a luciferase-mediatedreaction, and a derivative of luciferin which is a substrate for thenonluciferase enzyme, yielding a mixture. A reaction between thenonluciferase enzyme and the derivative yields a luminogenic productthat is a substrate for the luciferase. In one embodiment, thederivative is a compound of formula I. In another embodiment, thederivative is a compound of formula II. Luminescence in the mixture isdetected or determined, thereby detecting or determining the presence oramount of a molecule for the nonluciferase-mediated reaction in thesample.

The invention further provides a method to detect or determine thepresence or amount of a molecule for a luciferase-mediated reaction in asample. The method includes contacting a sample, a reaction mixture fora beetle luciferase, and a derivative of luciferin which is a substratefor the luciferase, to yield a reaction.

The invention also provides a method to detect the presence or amount ofa molecule in a sample. The method includes contacting a sample, a firstreaction mixture for a nonenzyme-mediated reaction and a derivative ofluciferin which in the presence of the molecule yields a luminogenicproduct that is a substrate for a luciferase, and then contacting atleast a portion of the first reaction and a second reaction mixture fora luciferase-mediated reaction, to yield a second reaction. Luminescencein the second reaction is detected or determined, thereby detecting ordetermining the presence or amount of the molecule. For instance, amixture is provided having a sample, a first reaction mixture for anonenzyme-mediated reaction and a derivative of luciferin which in thepresence of the molecule yields a a luminogenic product that is asubstrate for a beetle luciferase. At least a portion of the firstmixture and a second reaction mixture for a beetle luciferase-mediatedreaction are mixed, to yield a second mixture, and then luminescence inthe second mixture is detected or determined, thereby detecting ordetermining the presence or amount of the molecule.

For the biolumingenic assays described herein which employ luciferinderivatives with a lower background, those assays can use lower (orhigher) amounts of the derivative, and those derivatives may haveimproved reactivity, e.g., with a nonluciferase enzyme. In addition, forany of the bioluminogenic assays described herein, other reagents may beadded to reaction mixtures, including but not limited to those thatinhibit or prevent inactivation of luciferase, or otherwise extend orenhance luminescent signal.

Also provided is a method to identify or measure the potency of amodulator of a nonluciferase enzyme-mediated reaction. The methodincludes contacting one or more agents, a first reaction mixture for anonluciferase enzyme-mediated reaction, and a derivative of luciferinwhich is a substrate for the nonluciferase enzyme, so as to yield afirst mixture, or providing such a mixture, wherein the derivativeincludes an A or B ring modification relative to D-luciferin. The firstmixture in the absence of the one or more agents includes a luminogenicproduct that is a substrate for a beetle luciferase. At least a portionof the first mixture and a second reaction mixture for a beetleluciferase-mediated reaction are mixed, so as to yield a second mixture.Luminescence in the second mixture is compared with a control mixture,thereby identifying whether one or more of the agents modulates thenonluciferase enzyme-mediated reaction and/or to what extent and withwhat potency.

In one embodiment of the invention, test compounds can be screened andevaluated for their activities as substrates or cofactors of, orregulators, either inhibitors or activators, of an enzymatic ornonenzymatic reaction by using the luciferin and fluorophore derivativesof the present invention. A candidate compound may be determined to beregulator or a substrate of a reaction by contacting a reaction mixturewith a derivative and the test compound, under conditions that would, inthe absence of the test compound, yield bioluminescence, fluorescence,or a bioluminogenic product.

In one aspect of the invention, a method is provided to distinguishbetween a substrate and an inhibitor of a reaction. For example, thecompound is incubated with at least one enzyme under conditions whichallow for metabolism of the compound prior to providing a luciferinderivative under conditions that, in the absence of an inhibitor orsubstrate of the enzyme, would be suitable for interaction between theluciferin derivative and the enzyme. In one embodiment, the product ofthat reaction is a substrate of luciferase and in the presence ofluciferase yields a light emitting second reaction. The resulting lightemitting reaction is compared to the one obtained from contacting theenzyme with the compound and the derivative, under conditions thatwould, in the absence of an inhibitor of the enzyme, be suitable forinteraction between the luciferin derivative and the enzyme. Metabolismof the compound by the enzyme reduces its concentration in the assaymedium and may lead to an apparent loss of inhibitory activity comparedto conditions without metabolism of the compound which would indicate itwas a substrate for the enzyme. An inhibitory compound that was notmetabolized would show equal potency, irrespective of the time ofaddition of the substrate.

In one aspect of the invention, the compound is preferably contactedfirst with the enzyme for a first predetermined time period. Thereafter,the mixture is contacted with a luciferin derivative and bioluminescentenzyme, e.g., luciferase, simultaneously or contemporaneously, and themixture is allowed to incubate for a second predetermined time period.

In another aspect of the invention, the compound is incubated with theenzyme for a first predetermined time period to form a first mixture.Thereafter, the first mixture is contacted with the luciferinderivative, to form a second mixture that is allowed to incubate for asecond predetermined time period. The second mixture is then contactedwith a bioluminescent enzyme, e.g., luciferase, to form a third mixture,which is allowed to incubate for a third predetermined time period.Thereafter, the activity resulting from the interaction of the enzymewith the compound is determined by measuring luminescence during and/orafter the third predetermined time period relative to a control (e.g.,no compound) reaction. In this way, for example, mechanism basedinhibitors of the first enzyme can be identified and distinguished fromnonmechanism based inhibitors because the first incubation with the testcompound but without the luciferin derivative will lead to a moreprofound inhibition by a mechanism based inhibitor than would beobserved without the first incubation or substrates of the firstreaction will show reduced inhibition.

In another embodiment of the invention, a cell-based method is providedfor screening a compound to determine its effect on enzyme activity ofthe cell. The test compound is contacted with a cell having the enzyme,either naturally or via recombinant expression, the luciferinderivative, and bioluminescent enzyme, e.g., luciferase, or contactedwith a cell having the enzyme and luciferase, and the derivative, for apredetermined period of time. Thus, in one embodiment, a cell thateither transiently or stably expresses a recombinant enzyme such as abioluminescent enzyme, e.g., luciferase, may be employed. Anyconventional method for creating transient or stable transfected cellsmay be used. In one embodiment, a luciferin derivative is contacted withand diffuses into a cell and, if the appropriate molecule is present,yields a product, which is a substrate for luciferase. If a luciferaseis present in the cell, luminescence can be detected. Alternatively, ina cell which lacks luciferase, the product passes out of the cell intothe medium and that medium is added to a luciferase reaction mixture.Thereafter, the activity resulting from the interaction of the cell withthe compound is determined by measuring luminescence of the reactionmixture relative to a control (minus test compound) reaction mixture.

In one aspect of the invention, the compound is preferably contactedfirst with the cell for a predetermined time period. Thereafter, thecell is contacted with the luciferin derivative and luciferasesimultaneously or contemporaneously and the mixture allowed to incubatefor a second predetermined time period. Enzyme activity is determined bymeasuring the amount of luminescence generated from the reaction mixturerelative to a control reaction mixture (e.g., minus test compound). Inanother aspect of the invention, the test compound is preferablycontacted first with the cell for a predetermined time period.Thereafter, the exposed cell is then contacted with the luciferinderivative and incubated for a second predetermined time period. Thecell is then contacted with luciferase to form a third mixture which isallowed to incubate for a third predetermined time period. Thereafter,the activity of the cell resulting from the interaction of the cell withthe test compound(s) is determined by measuring luminescence of thereaction mixture relative to a control reaction mixture (e.g., minustest compound). Detergent addition can rupture the cells and releasecell content.

A cell-based luminescence detection assay for molecules present in thecell medium, e.g., molecules which actively or via inactive mechanismsare present in the cell medium, can include adding a reaction mixturewith the luciferin derivative to the cell medium, or adding the cellmedium to a reaction mixture with the luciferin derivative, anddetecting luminescence.

In yet another embodiment of the cell-based assay of the invention, thecells may be lysed in an appropriate lysis buffer. For animal cells, abuffer with 0.1-1.0% non-ionic detergents such as Triton X 100 orTergitol is typically sufficient. Bacteria, plant, fungal or yeast cellsare usually more difficult to lyse. Detergents, freeze/thaw cycles,hypotonic buffers, sonication, cavitation or combinations of thesemethods may be used. The method of lysis that produces a lysate iscompatible with luciferase or other enzyme activity, or the detection ofother molecules or conditions.

The presence or activity of nonluciferase enzymes may be measured incells grown in culture medium or in cells within animals, e.g., livinganimals. For measurements in cells in animals, a luciferin derivativemay be administered to the animal, e.g., injected into the animal oradded to an aqueous solution, e.g., water, or food consumed by theanimal. Conversion of the derivative to a product that is a luciferasesubstrate may be detected by luminescence mediated by luciferaseexpressed in cells in the animal, e.g., transgenic cells, by luciferaseadministered to the animal, e.g., injected into the animal, or bycollecting physiological fluids, e.g., blood, plasma, urine, and thelike, or tissue samples, and combining those with a luciferase reagent.

In one embodiment, the derivative employed in the methods is notaminoluciferin which is modified to include a protease substrate via apeptide bond at the amino group which optionally also has a protectedcarboxyl group at position 5 in the C ring, or any of luciferin 6′methyl ether, luciferin 6′ chloroethyl ether, 6′ deoxyluciferin, 6′luciferin 4-trifluoromethyl benzylether, luciferin 6′ phenylethylether,luciferin 6′ geranyl ether, luciferin 6′ ethyl ether, 6′ luciferinprenyl ether, 6′ luciferin 2-picolinylether, 6′ luciferin3-picolinylether, 6′ luciferin 4-picolinyl ether, luciferin 6′ benzylether, D-luciferin-O-β-galactopyranoside, D-luciferin-O-sulfate,D-luciferin-O-phosphate, D-luciferyl-L-phenylalanine, andD-luciferyl-L-N^(α)-arginine. In one embodiment, the derivative employedin the methods is not a luciferin derivative disclosed in U.S. publishedapplication 20040171099, the disclosure of which is incorporated byreference herein.

Assays which employ two reactions may be conducted simultaneously (onestep) or sequentially (two step) to detect one or more moietiesincluding proteins (peptides or polypeptides), e.g., enzymes,substrates, cofactors, inhibitors or activators for enzymatic reactions,or conditions, e.g., redox conditions. A sequential reaction may beconducted in the same vessel, e.g., a well of a multiwell plate. For atwo step assay, the first reaction mixture may contain all of thereagents or less than all of the reagents for a nonluciferaseenzyme-mediated reaction, where one of the reagents that is absent isthe one to be detected in a sample, e.g., a cell lysate. For instance, anonluciferase enzyme-mediated reaction is performed under conditionseffective to convert a luciferin derivative that is a substrate for thenonluciferase and a prosubstrate of luciferase, to a product that is asubstrate of luciferase. The first reaction may be quenched at the time,or prior to addition, of a luciferase reaction mixture. For instance, aquencher of the first reaction may be present in the luciferase reactionmixture. The luciferase reaction mixture preferably substantially lacksa substrate for the luciferase, e.g., the only source of substrate forthe luciferase is provided by a reaction between the nonluciferaseenzyme and the derivative. When all the reagents for the first reactionare present in the first reaction mixture, the assay may be employed toidentify moieties that alter the reaction, e.g., inhibitors or enhancersof the reaction. After performing the reactions, either simultaneouslyor sequentially, the presence or amount of one or more molecules, or oneor more inhibitors or activators of the reaction(s) is/are detected ordetermined and/or to what extent and with what potency.

For a one step assay, a reaction mixture may contain reagents for tworeactions, such as reagents for a nonluciferase enzyme-mediated reactionand a luciferase-mediated reaction or for a nonenzymatic reaction and aluciferase-mediated reaction, or a reaction mixture for a singlereaction, e.g., for a reaction between a derivative of a fluorophorewhich is a substrate for an enzyme and the enzyme or aluciferase-mediated reaction, e.g., a luciferase is suspected in asample to be tested.

For assays which employ two reactions, the order of adding the moleculesfor the assays can vary. If initiated and conducted sequentially(whether in the same vessel or not), adjustments to reaction conditions,e.g., reagent concentration, temperatures or additional reagents, may beperformed. For instance, a quenching agent or enhancing agent may beadded between reactions (see, e.g., U.S. Pat. Nos. 5,774,320 and6,586,196, the disclosures of which are specifically incorporated byreference herein). In one embodiment, the two or more reactions arecarried out simultaneously in a single reaction mixture. Optionally, theassays are a homogeneous assay, e.g., the components are mixed prior toadding the mixture to the sample. Results may be read without additionaltransfer of reagents.

The assays of the present invention thus allow the detection of one ormore molecules or conditions in a sample, e.g., a sample which includeseukaryotic cells, e.g., yeast, avian, plant, insect or mammalian cells,including but not limited to human, simian, murine, canine, bovine,equine, feline, ovine, caprine or swine cells, or prokaryotic cells, orcells from two or more different organisms, or cell lysates orsupernatants thereof, or a sample which includes a purified form of themolecule, e.g., purified nonluciferase enzyme which is useful to preparea standard curve. The cells may not have been genetically modified viarecombinant techniques (nonrecombinant cells), or may be recombinantcells which are transiently transfected with recombinant DNA and/or thegenome of which is stably augmented with a recombinant DNA, or whichgenome has been modified to disrupt a gene, e.g., disrupt a promoter,intron or open reading frame, or replace one DNA fragment with another.The recombinant DNA or replacement DNA fragment may encode a molecule tobe detected by the methods of the invention, a moiety which alters thelevel or activity of the molecule to be detected, and/or a gene productunrelated to the molecule or moiety that alters the level or activity ofthe molecule.

The present methods can be employed to detect a molecule for anenzyme-mediated reaction, a nonenzymatic-mediated reaction or condition.For instance, molecules or conditions to be detected by the methodinclude but are not limited to enzymes, e.g., demethylases, oxidases(e.g., a MAO), deacetylases, deformylases, proteases (proteosome,calpain, beta-secretase, cathepsin, calpain, thrombin, granzyme B),phosphatases, kinases, peroxidases, transferases, e.g., GST, sulfotases,beta-lactamases, cytochrome P450 enzymes, esterase, e.g.,acetylcholinesterase, dehydrogenase, luciferase, substrates, inhibitors,co-factors, activators of enzyme mediated reactions, reactive oxygenspecies, reducing conditions and transcriptional regulators orregulators of gene transcription. The enzymes employed in the methods,either enzymes to be detected or enzymes which are useful to detect asubstrate or cofactor, can be selected from any combination of enzymesincluding recombinant and endogenous (native) enzymes. In oneembodiment, the enzyme to be detected is an endogenous enzyme. Inanother embodiment, the enzyme is a recombinant enzyme. Othercombinations apparent to one of ordinary skill in the art can be used inthe present assays and methods according to the teachings herein. Theenzymes include but are not limited to proteases, phosphatases,peroxidases, sulfatases, peptidases, oxidases, dealkylases, deformylasesand glycosidases. The enzyme may be a hydrolase, oxidoreductase, lyase,transferase, e.g., glutathione S transferase, isomerase, ligase, orsynthase. Of particular interest are classes of enzymes that havephysiological significance. These enzymes include protein peptidases,esterases, protein phosphatases, glycosylases, proteases,dehydrogenases, oxidases, oxygenases, reductases, methylases and thelike. Exemplary cleavage sites for some proteases are set forth in FIG.2. Enzymes of interest include those involved in making or hydrolyzingesters, both organic and inorganic, glycosylating, and hydrolyzingamides. In any class, there may be further subdivisions.

In particular, enzymes that are useful in the present invention includeany protein that exhibits enzymatic activity, e.g., lipases,phospholipases, sulphatases, ureases, peptidases, proteases andesterases, including acid phosphatases, glucosidases, glucuronidases,galactosidases, carboxylesterases, and luciferases. In one embodiment,the enzyme is a hydrolytic enzyme. Examples of hydrolytic enzymesinclude alkaline and acid phosphatases, esterases, decarboxylases,phospholipase D, P-xylosidase, β-D-fucosidase, thioglucosidase,β-D-galactosidase, α-D-galactosidase, α-D-glucosidase, β-D-glucosidase,β-D-glucuronidase, α-D-mannosidase, β-D-mannosidase,β-D-fructofuranosidase, and β-D-glucosiduronase.

In one embodiment, the invention provides the use of a compound offormula III or IIIA as described herein.

In one embodiment, an enzyme, for instance a nonproteolytic enzyme, isdetected using a substrate which is covalently linked to a fluorophore.In one embodiment, the substrate includes a recognition site for theenzyme. In the absence of the appropriate enzyme or cofactor, a mixtureincluding such a substrate generates minimal light at the emissionwavelength as the fluorescent properties of the fluorophore arequenched, e.g., by the proximity of the quenching group. In the presenceof the appropriate enzyme, cleavage of the conjugate yields thefluorophore.

III. Luciferin Derivatives

In one embodiment, derivatives of luciferin or aminoluciferin have thefollowing structure: L-X-M-Y—R (compound of formula IV), wherein L, ifpresent, may be a substrate for an enzyme or another molecule whichinteracts with the enzyme; X may be O, NH, or a linker, e.g., aself-cleavable linker which spontaneously cleaves to yield M-Y—R after Lhas been removed from L-X-M-Y—R; M may be luciferin, quinolinylluciferin or naphthyl luciferin (X═O), or aminoluciferin oraminoquinolinyl luciferin (X═NH); Y is O (ester), NH (amide), NH—NH(hydrazide), or S (thioester); and R, if present, may be alkyl, anaromatic molecule, a peptide, an oligonucleotide, or a self-cleavablelinker attached to a substrate for an enzyme. In one embodiment, thederivative may be modified at L or R to include a substrate for anenzyme such as a P450 enzyme, protease, MAO, FMO, or GST. In oneembodiment, a derivative of the invention is a substrate for luciferase,including 6-aminoquinolinyl luciferin, which is a substrate ofluciferase having substantial light output.

Bioluminescent substrates according to the present invention arederivatives of luciferin or aminoluciferin, i.e., are luminescentsubstrates other than luciferin or aminoluciferin, and include compoundshaving the general formulas described below including, e.g., formulas Iand II.

In one embodiment, the invention provides a compound of formula I:

wherein

Y is N,N-oxide, N—(C₁-C₆)alkyl, or CH;

when Y is N, then X is not S;

X is S, O, CH═CH, N═CH, or CH═N;

when X is S, then Y is not N;

Z and Z′ are independently H, OR, NHR, or NRR;

Z″ is O, S, NH, NHR, or N═N;

Q is carbonyl or CH₂;

W¹ is H, halo, (C₁-C₆)alkyl, (C₂-C₂₀)alkenyl, hydroxyl, or(C₁-C₆)alkoxy; or

W¹ and Z are both keto groups on ring A, and at least one of the dottedlines denoting optional double bonds in ring A is absent;

each W² is independently H, halo, (C₁-C₆)alkyl, (C₂-C₄)alkenyl,hydroxyl, or (C₁-C₆)alkoxy;

each of K¹, K², K³, and K⁴ are independently CH, N,N-oxide, orN—(C₁-C₆)alkyl, and the dotted lines between K¹ and K², and K³ and K⁴,denote optional double bonds;

A′ and B′ are optional aromatic rings fused to ring A, only one of whichis present in the compound, so as to form a fused tricyclic system; and

-   -   when B′ is present, the group Z is present, and    -   when A′ is present, the group Z is absent; and

the dotted line in ring B is an optional double bond;

each R is independently H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₁₂)alkoxy, (C₆-C₃₀)aryl,heteroaryl, heterocycle, (C₁-C₂₀)alkylsulfoxy, (C₆-C₃₀)arylsulfoxy,heteroarylsulfoxy, (C₁-C₂₀)alkylsulfonyl, (C₆-C₃₀)arylsulfonyl,heteroarylsulfonyl, (C₁-C₂₀)alkylsulfinyl, (C₆-C₃₀)arylsulfinyl,heteroarylsulfinyl, (C₁-C₂₀)alkoxycarbonyl, amino, NH(C₁-C₆)alkyl,N((C₁-C₆)alkyl)₂, tri(C₁-C₂₀)ammonium(C₁-C₂₀)alkyl,heteroaryl(C₁-C₂₀)alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen, (C₆-C₃₀)arylthio,(C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, (C₆-C₃₀)arylphosphate,(C₆-C₃₀)arylphosphonate, phosphate, sulfate, saccharide, or M⁺optionally when Z″ is oxygen, wherein M is an alkali metal;

or when Z or Z′ is NR¹R¹, R¹R¹ together with the N to which they areattached forms a heteroaryl or heterocycle group;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, amino, aryl,heteroaryl, or heterocycle group is optionally substituted with 1, 2, 3,4, or 5 substituents selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxyl,(C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —COOR^(x),—SO₂R^(x), —SO₃R^(x), nitro, amino, (C₁-C₂₀)alkyl-S(O)—,(C₁-C₂₀)alkyl-SO₂—, phosphate, (C₁-C₂₀)alkylphosphate,(C₁-C₂₀)alkylphosphonate, NH(C₁-C₆)alkyl, NH(C₁-C₆)alkynyl,N((C₁-C₆)alkyl)₂, N((C₁-C₆)alkynyl)₂, mercapto, (C₁-C₂₀)alkylthio,(C₆-C₃₀)aryl, (C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, andheterocycle, and each substituent is optionally substituted with one tothree R groups;

R^(x) is H, (C₁-C₆)alkyl, or (C₆-C₃₀)aryl;

when Z or Z′ comprises a nitrogen moiety, one or both of the hydrogensof the Z or Z′ nitrogen moiety may be replaced by (C₁-C₂₀)alkyl or thegroup L, wherein L is an amino acid radical, a peptide radical having upto 20 amino acid moieties, or any other small molecule that is asubstrate for a nonluciferase; with the proviso that when L is an aminoacid radical or a peptide radical, at least one W² is not H;

when Z is a hydroxyl group or a nitrogen moiety, H of the hydroxyl ornitrogen moiety may be replaced by (HO)₂P(O)—OCH₂—, sulfo, —PO₃H₂, or bya cephalosporanic acid attached to the group Z via a carbon chain of oneto about 12 carbon atoms; with the proviso that when ring B is athiazole ring, the sulfo or the —PO₃H₂ group is attached to the hydroxyloxygen via a (C₁-C₆)alkylene group;

when Z or Z′ is a hydroxyl group or a nitrogen moiety, or when Z″—R is ahydroxyl group, one H of the hydroxyl or nitrogen moiety may be replacedby the group L′-linker, wherein L′ is a group removable by an enzyme tofree the linker, and linker is a carbon chain that can self-cleave,optionally interrupted by one or more nitrogen atoms, oxygen atoms,carbonyl groups, optionally substituted aromatic rings, or peptidebonds,

linker is attached to L′ via an oxygen atom or an NH group at oneterminus of the linker and the other terminus of the linker forms anether, ester, or amide linkage with a group Z, Z′, or Z″—R;

when Z is OR, formula I is optionally a dimer connected at the two Arings via a linker comprising a (C₁-C₁₂)alkyl diradical that isoptionally interrupted by one to four O atoms, N atoms, or an optionallysubstituted aryl, heteroaryl, or heterocycle group to form a bridgebetween the dimer of formula I, and the R group of each Z groupconnecting the dimer of formula I is replaced by the bridge;

A⁻ is an anion, present when a quaternary nitrogen is present;

or a salt thereof;

provided that:

when rings A and B form a naphthalene or quinoline ring system, then W¹is not hydrogen;

when a ring A substituent is OH, then -Q-Z″—R is not —C(O)—NH—NH₂;

when Y is N or CH and X is CH═CH and W¹ is H, then Z is not OH attachedto K³; and

when Y is N or CH and X is CH═CH and Z is H, then W¹ is not OH attachedto K³.

In another embodiment, the invention provides a compound of formula IA:

wherein

Y is N,N-oxide, N—(C₁-C₆)alkyl, or CH;

when Y is N, then X is not S;

X is S, O, CH═CH, N═CH, or CH═N;

when X is S, then Y is not N;

Z is H, OR, NHR, or NRR;

Z″ is O, S, NH, NHR, or N═N;

W¹ is H, halo, hydroxyl, (C₁-C₆)alkyl, or (C₁-C₆)alkoxy;

the dotted line in ring B is an optional double bond;

each R is independently H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₁₂)alkoxy, (C₆-C₃₀)aryl,heteroaryl, heterocycle, (C₁-C₂₀)alkylsulfoxy, (C₆-C₃₀)arylsulfoxy,heteroarylsulfoxy, (C₁-C₂₀)alkylsulfonyl, (C₆-C₃₀)arylsulfonyl,heteroarylsulfonyl, (C₁-C₂₀)alkylsulfinyl, (C₆-C₃₀)arylsulfinyl,heteroarylsulfinyl, (C₁-C₂₀)alkoxycarbonyl, amino, NH(C₁-C₆)alkyl,N((C₁-C₆)alkyl)₂, tri(C₁-C₂₀)ammonium(C₁-C₂₀)alkyl,heteroaryl(C₁-C₂₀)alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen, (C₆-C₃₀)arylthio,(C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, (C₆-C₃₀)arylphosphate,(C₆-C₃₀)arylphosphonate, phosphate, sulfate, saccharide, or M⁺optionally when Z″ is oxygen, wherein M is an alkali metal;

or when Z or Z′ is NR¹R¹, R¹R¹ together with the N to which they areattached forms a heteroaryl or heterocycle group;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, amino, aryl,heteroaryl, or heterocycle group is optionally substituted with 1, 2, 3,4, or 5 substituents selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxyl,(C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —COOR^(x),—SO₂R^(x), —SO₃R^(x), nitro, amino, (C₁-C₂₀)alkyl-S(O)—,(C₁-C₂₀)alkyl-SO₂—, phosphate, (C₁-C₂₀)alkylphosphate,(C₁-C₂₀)alkylphosphonate, NH(C₁-C₆)alkyl, NH(C₁-C₆)alkynyl,N((C₁-C₆)alkyl)₂, N((C₁-C₆)alkynyl)₂, mercapto, (C₁-C₂₀)alkylthio,(C₆-C₃₀)aryl, (C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, andheterocycle, and each substituent is optionally substituted with one tothree R groups;

R^(x) is H, (C₁-C₆)alkyl, or (C₆-C₃₀)aryl;

A⁻ is an anion, present when a quaternary nitrogen is present;

or a salt thereof;

provided that:

when rings A and B form a naphthalene or quinoline ring system, then W¹is not hydrogen;

when a ring A substituent is OH, then -Q-Z″—R is not —C(O)—NH—NH₂;

when Y is N or CH and X is CH═CH and W¹ is H, then Z is not OH attachedto carbon-6 of ring A; and

when Y is N or CH and X is CH═CH and Z is H, then W¹ is not OH attachedto carbon-6 of ring A.

As illustrated by formulas I and IA, the core structure of rings A and Bcan be a number of different ring systems. Modification of ring B allowsthe core structure to include benzofuran, benzothiophene, benzoxazole,naphthalene, quinoline, isoquinoline, quinazoline, and quinoxyline ringsystems, including the corresponding N-oxide and N-alkyl derivatives.Ring B modification also allows for access to N-oxide and N-alkylderivatives of benzo[d]thiazole. Furthermore, by substituting a carbonatom of ring A in formula I with N,N-oxide, or N-alkyl (e.g.,substituting one value of K¹, K², K³, or K⁴ for another value), otherring systems can be obtained, such as 1,8-naphthyridine,1,7-naphthyridine, 1,6-naphthyridine, and 1,5-naphthyridine ringsystems; pyrido[3,2-b]pyrazine, pyrido[4,3-b]pyrazine,pyrido[3,4-b]pyrazine, and pyrido[2,3-b]pyrazine ring systems;pyrido[2,3-d]pyrimidine, pyrido[3,4-d]pyrimidine,pyrido[4,3-d]pyrimidine, and pyrido[3,2-d]pyrimidine ring systems;benzo[d]oxazole, oxazolo[4,5-b]pyridine, oxazolo[4,5-c]pyridine,oxazolo[5,4-c]pyridine, and oxazolo[5,4-b]pyridine ring systems; andthiazolo[4,5-b]pyridine, thiazolo[4,5-c]pyridine,thiazolo[5,4-c]pyridine, and thiazolo[5,4-b]pyridine ring systems.

Formula I also illustrates that by substituting a second carbon atom ofring A with N,N-oxide, or N-alkyl, the corresponding pyrazine,pyramidine, and pyridazine ring A analogs can be obtained for each ofthe above described ring systems. The substitution of a third carbonatom in ring A of formula I with N,N-oxide, or N-alkyl provides accessto the corresponding 1,2,4-triazine ring system derivatives.

Formulas I and IA further include the various dihydro, tetrahydro, andhexahydro derivatives of each of its ring systems.

In another embodiment, the invention provides a compound of formula II:

wherein

Z and Z′ are independently OR¹, NHR¹, or NR¹R¹;

Z″ is O, S, NH, NHR, or N═N;

Q is carbonyl or CH₂;

W¹ is H, halo, (C₁-C₆)alkyl, (C₂-C₂₀)alkenyl, hydroxyl, or(C₁-C₆)alkoxy; or

W¹ and Z are both keto groups on ring A, and at least one of the dottedlines denoting optional double bonds in ring A is absent;

each W² is independently H, halo, (C₁-C₆)alkyl, (C₂-C₄)alkenyl,hydroxyl, or (C₁-C₆)alkoxy;

each of K¹, K², K³, and K⁴ are independently CH, N,N-oxide, orN—(C₁-C₆)alkyl, and the dotted lines between K¹ and K², and K³ and K⁴,denote optional double bonds;

A′ and B′ are optional aromatic rings fused to ring A, only one of whichis present in the compound, so as to form a fused tricyclic system; and

-   -   when B′ is present, the group Z is present, and    -   when A′ is present, the group Z is absent; and

R is H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₃-C₂₀)cycloalkyl, (C₁-C₁₂)alkoxy, (C₆-C₃₀)aryl, heteroaryl,heterocycle, (C₁-C₂₀)alkylsulfoxy, (C₆-C₃₀)arylsulfoxy,heteroarylsulfoxy, (C₁-C₂₀)alkoxycarbonyl, amino, NH(C₁-C₆)alkyl,N((C₁-C₆)alkyl)₂, tri(C₁-C₂₀)ammonium(C₁-C₂₀)alkyl,heteroaryl(C₁-C₂₀)alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen, saccharide, or M⁺optionally when Z″ is oxygen, wherein M is an alkali metal;

R¹ is (C₆-C₃₀)aryl, heteroaryl, heterocycle, (C₁-C₂₀)alkylthio,(C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂, —SO₃(C₁-C₂₀)alkyl, saccharide,(C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, (C₆-C₃₀)arylthio,(C₆-C₃₀)aryl-S(O)—, (C₆-C₃₀)aryl-SO₂, —SO₃(C₆-C₃₀)aryl,(C₆-C₃₀)arylphosphate, (C₆-C₃₀)arylphosphonate, or R¹ is (C₁-C₂₀)alkylsubstituted by R²;

R² is (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxyl, (C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, hydroxyl,—COOR^(x), —SO₃R^(x), (C₁-C₂₀)alkylthio, (C₆-C₃₀)arylthio,(C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂—, nitro, amino, NH(C₁-C₆)alkyl,NH(C₁-C₆)alkynyl, N((C₁-C₆)alkyl)₂, or N((C₁-C₆)alkynyl)₂, mercapto,saccharide, or trifluoromethyl;

or when Z or Z′ is NR¹R¹, R¹R¹ together with the N to which they areattached forms a heteroaryl or heterocycle group;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, amino, aryl,heteroaryl, or heterocycle group is optionally substituted with 1, 2, 3,4, or 5 substituents selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxyl,(C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —COOR^(x),—SO₂R^(x), —SO₃R^(x), (C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂—,phosphate, (C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, nitro,amino, NH(C₁-C₆)alkyl, NH(C₁-C₆)alkynyl, N((C₁-C₆)alkyl)₂,N((C₁-C₆)alkynyl)₂, mercapto, (C₁-C₂₀)alkylthio, (C₆-C₃₀)aryl,(C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, and heterocycle, andeach substituent is optionally substituted with one to three R groups;

R^(x) is H or (C₁-C₆)alkyl;

when Z or Z′ comprises a nitrogen moiety, a hydrogen of the Z or Z′nitrogen moiety may be replaced by the group L, wherein L is an aminoacid radical, a peptide radical having up to 20 amino acid moieties, orany other small molecule that is a substrate for a nonluciferase; withthe proviso that when L is an amino acid radical or a peptide radical,at least one of W¹ or a W² is not H;

when Z is a hydroxyl group or a nitrogen moiety, H of the hydroxyl ornitrogen moiety may be replaced by (HO)₂P(O)—OCH₂—, sulfo, —PO₃H₂, or bya cephalosporanic acid attached to the group Z via a carbon chain of oneto about 12 carbon atoms; with the proviso that the sulfo or the —PO₃H₂group is attached to the hydroxyl oxygen via a (C₁-C₆)alkylene group;

when Z or Z′ is a hydroxyl group or a nitrogen moiety, or when Z″—R is ahydroxyl group, one H of the hydroxyl or nitrogen moiety may be replacedby the group L′-linker, wherein L′ is a group removable by an enzyme tofree the linker, and linker is a carbon chain that can self-cleave,optionally interrupted by one or more nitrogen atoms, oxygen atoms,carbonyl groups, optionally substituted aromatic rings, or peptidebonds,

linker is attached to L′ via an oxygen atom or an NH group at oneterminus of the linker and the other terminus of the linker forms anether, ester, or amide linkage with a group Z, Z′, or Z″—R;

when Z is OR¹, formula II is optionally a dimer connected at the two Arings via linker comprising a (C₁-C₁₂)alkyl diradical that is optionallyinterrupted by one to four O atoms, N atoms, or an optionallysubstituted aryl, heteroaryl, or heterocycle group to form a bridgebetween the dimer of formula II, and the R¹ group of each Z groupconnecting the dimer of formula II is replaced by the bridge;

provided that a saccharide is not directly attached to K³;

A⁻ is an anion, present when a quaternary nitrogen is present;

or a salt thereof.

In yet another embodiment, the invention provides a compound of formulaIIA:

wherein

Z is OR¹, NHR¹, or NR¹R¹;

Z″ is O, S, NH, NHR, or N═N;

W¹ is H, halo, hydroxyl, (C₁-C₆)alkyl, or (C₁-C₆)alkoxy;

R is H, (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₃-C₂₀)cycloalkyl, (C₁-C₁₂)alkoxy, (C₆-C₃₀)aryl, heteroaryl,heterocycle, (C₁-C₂₀)alkylsulfoxy, (C₆-C₃₀)arylsulfoxy,heteroarylsulfoxy, (C₁-C₂₀)alkoxycarbonyl, amino, NH(C₁-C₆)alkyl,N((C₁-C₆)alkyl)₂, tri(C₁-C₂₀)ammonium(C₁-C₂₀)alkyl,heteroaryl(C₁-C₂₀)alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen, saccharide, or M⁺optionally when Z″ is oxygen, wherein M is an alkali metal;

R¹ is (C₆-C₃₀)aryl, heteroaryl, heterocycle, (C₁-C₂₀)alkylthio,(C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂, —SO₃(C₁-C₂₀)alkyl, saccharide,(C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, (C₆-C₃₀)arylthio,(C₆-C₃₀)aryl-S(O)—, (C₆-C₃₀)aryl-SO₂, —SO₃(C₆-C₃₀)aryl,(C₆-C₃₀)arylphosphate, (C₆-C₃₀)arylphosphonate, or R¹ is (C₁-C₂₀)alkylsubstituted by R²;

R² is (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl,(C₁-C₂₀)alkoxyl, (C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, hydroxyl,—COOR^(x), —SO₃R^(x), (C₁-C₂₀)alkylthio, (C₆-C₃₀)arylthio,(C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂—, nitro, amino, NH(C₁-C₆)alkyl,NH(C₁-C₆)alkynyl, N((C₁-C₆)alkyl)₂, or N((C₁-C₆)alkynyl)₂, mercapto,saccharide, or trifluoromethyl;

or when Z or Z′ is NR¹R¹, R¹R¹ together with the N to which they areattached forms a heteroaryl or heterocycle group;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, amino, aryl,heteroaryl, or heterocycle group is optionally substituted with 1, 2, 3,4, or 5 substituents selected from (C₁-C₂₀)alkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxyl,(C₁-C₂₀)alkylcarbonyl, (C₁-C₂₀)alkylcarboxyl, halo, hydroxyl, —COOR^(x),—SO₂R^(x), —SO₃R^(x), (C₁-C₂₀)alkyl-S(O)—, (C₁-C₂₀)alkyl-SO₂—,phosphate, (C₁-C₂₀)alkylphosphate, (C₁-C₂₀)alkylphosphonate, nitro,amino, NH(C₁-C₆)alkyl, NH(C₁-C₆)alkynyl, N((C₁-C₆)alkyl)₂,N((C₁-C₆)alkynyl)₂, mercapto, (C₁-C₂₀)alkylthio, (C₆-C₃₀)aryl,(C₆-C₃₀)arylthio, trifluoromethyl, ═O, heteroaryl, and heterocycle, andeach substituent is optionally substituted with one to three R groups;

R^(x) is H or (C₁-C₆)alkyl;

when Z is OR¹, formula IIA is optionally a dimer connected at the two Arings via linker comprising a (C₁-C₁₂)alkyl diradical that is optionallyinterrupted by one to four O atoms, N atoms, or an optionallysubstituted aryl, heteroaryl, or heterocycle group to form a bridgebetween the dimer of formula IIA, and the R¹ group of each Z groupconnecting the dimer of formula II is replaced by the bridge;

provided that a saccharide is not directly attached to K³;

A⁻ is an anion, present when a quaternary nitrogen is present;

or a salt thereof.

As illustrated by formulas II and IIA, the core structure of rings A andB can also be a variety of ring systems. In addition tobenzo[d]thiazole, substituting a carbon atom of ring A of formula IIwith N,N-oxide, or N-alkyl allows access to thiazolo[4,5-b]pyridine,thiazolo[4,5-c]pyridine, thiazolo[5,4-c]pyridine, andthiazolo[5,4-b]pyridine ring systems, and their corresponding N-oxidesand N-alkyl derivatives.

Formula II also illustrates that by substituting a second carbon atom ofring A with N,N-oxide, or N-alkyl, the corresponding pyrazine,pyramidine, and pyridazine ring A analogs can be obtained for each ofthe above described ring systems. The substitution of a third carbonatom in ring A of formula II provides access to the corresponding1,2,4-triazine derivatives.

Formulas II and IIA include the various dihydro and tetrahydroderivatives of each of its ring systems.

All ring systems described above can be substituted as illustrated inthe formulas described herein, for example, formulas I and/or II. Eachof the above-described individual ring systems and their derivatives,substituted as described in the formulas herein, are separateembodiments of the invention.

Other deriviates and their use in luminogenic assays is describedhereinbelow.

The use of the luciferin derivatives described herein can result in anassay which produces a measurable change in optical properties uponinteraction with a nonluciferase molecule, which interaction may alterthe structure of the luciferin derivative. As described herein, theproduct of a reaction between a luciferin derivative and a nonluciferaseenzyme or other molecule of interest need not be D-luciferin oraminoluciferin. For example, a luciferin derivative may include asubstrate that includes a reactive chemical group for a nonluciferaseenzyme linked to luciferin or aminoluciferin via a chemical linker.Transformation of the reactive chemical group of the derivative by thenonluciferase enzyme may yield a product that contains (retains) aportion of the substrate, a portion of the chemical linker, the chemicallinker, or a portion of the substrate and the chemical linker, and thatproduct is a substrate for luciferase. Also provided are luciferinderivatives which, after interaction with a nonluciferase enzyme orother molecule, may yield a product that optionally undergoes one ormore further reactions, e.g., β-elimination, to yield a suitablesubstrate for luciferase. Luciferin derivatives in which the backbone ofluciferin is further modified in its ring structure, e.g., a quinolyl ornapthyl luciferin, are provided, as well as advantageously providingmodifications at the carboxy position of the thiazole ring, to provideimproved characteristics to the luciferin derivative. Derivatives withcertain modifications provide for or improve assays for certainnonluciferase enzymes or molecules. For instance, as describedhereinbelow, a pH insensitive derivative of luciferin was identifiedthat is useful in biological assays that may be run at a pH other thanphysiological pH, i.e., less than about pH 7.0 and greater than about pH7.8. Thus, bioluminescent methods that employ a luciferin derivative ofthe invention may be used to detect one or more molecules, e.g., anenzyme, a cofactor for an enzymatic reaction such as ATP, an enzymesubstrate, an enzyme inhibitor, an enzyme activator, or OH radicals, orone or more conditions, e.g., redox conditions.

In one embodiment, the invention provides a compound of formula (V):

wherein

Y is N,N-oxide, N-loweralkyl, or CH;

X is S, CH═CH, or N═C,

Z and Z′ are H, OR, NHR, or NRR;

Z″ is O, S, NH, NHR, or N═N;

each W is independently H, halo, C₁₋₆alkyl, C₂₋₂₀alkenyl, hydroxyl, or

C₁₋₆alkoxy; or

W and Z on ring A are both keto groups;

each of K¹, K², K³, and K⁴ are independently CH, N,N-oxide, orN-loweralkyl;

R is H, C₁₋₂₀alkyl, substituted C₁₋₂₀alkyl, C₂₋₂₀alkenyl, substitutedC₂₋₂₀alkenyl, halogenated C₂₋₂₀alkenyl, substituted halogenatedC₂₋₂₀alkenyl, C₃₋₂₀alkynyl, substituted C₃₋₂₀alkynyl,C₂₋₂₀alkenylC₁₋₂₀alkyl, substituted C₂₋₂₀alkenylC₁₋₂₀alkyl,C₃₋₂₀alkynylC₂₋₂₀alkenyl, substituted C₃₋₂₀alkynylC₂₋₂₀alkenyl,C₃₋₂₀cycloalkyl, substituted C₃₋₂₀cycloalkyl, C₆₋₃₀aryl, heteroaryl,C₆₋₃₀arylC₁₋₂₀alkyl, substituted C₆₋₃₀aryl, substituted heteroaryl,substituted C₆₋₃₀arylC₁₋₂₀alkyl, alkylsulfoxyC₁₋₂₀alkyl,C₁₋₂₀alkoxycarbonyl, C₆₋₃₀arylC₁₋₂₀alkoxycarbonyl,C₆₋₃₀arylthioC₁₋₂₀alkyl, hydroxyC₁₋₂₀alkyl, triC₁₋₂₀ammoniumC₁₋₂₀alkyl,heteroarylC₁₋₂₀alkyl, substituted heteroarylC₁₋₂₀alkyl, heteroarylhaving quaternary nitrogen, heteroarylcarbonyl having quaternarynitrogen, and N-methyl-tetrahydropyridinyl; and M⁺ when Z″ is oxygen,wherein M is an alkali metal; wherein the alkyl, cycloalkyl, alkenyl,and/or alkynyl groups may be optionally substituted by one moreC₁₋₂₀alkyl, halo, hydroxyl, acetyl, amino, lower alkylamino, loweralkynylamino, imidazolinylmethylamino, di-lower alkylamino, di-loweralkynylamino, piperidino, pyrrolidino, azetidino, aziridino,

di-imidazolinylmethylamino, mercapto, C₁₋₂₀alkylthio, C₆₋₃₀arylthio, ortrifluoromethyl groups, substituted C₆₋₃₀arylC₁₋₂₀alkyl carbonyl; andeach group R is defined independently if more than one is present;

Q is (C═O)_(n) or (CH₂)_(n);

n is 0, 1, or 2;

and wherein

when Z or Z″ is amino, one or both of the hydrogens may be replaced byC₁₋₂₀alkyl, or the group L, wherein

L is an amino acid radical, a peptide radical having up to 20 amino acidmoieties, or may be any other small molecule that is a substrate for anonluciferase; with the proviso that when L is an amino acid radical ora peptide radical, W is not H;

and wherein

when Z is hydroxyl or amino, H may be replaced by (HO)₂P(O)—OCH₂—,sulfo,

or —PO₃H₂, or by cephalosporanic acid attached to the group Z via acarbon chain of one or more carbon atoms; with the proviso that whenring B is a thiazole ring, the sulfo or the —PO₃H₂ group is attached tothe hydroxyl oxygen via a loweralkylene chain; and

when Z is hydroxyl or amino or when Z″—R is hydroxyl, H may be replacedby the group L′-linker, wherein L′ is a group removable by an enzyme tofree the linker, and the linker is a carbon chain that may optimallyself-cleave, optionally interrupted by one or more nitrogen atoms,oxygen atoms, carbonyl groups, (substituted)aromatic rings, or peptidebonds, and

linker is attached to L′ via an oxygen atom or an NH group at oneterminus of the linker and the other terminus of the linker forms anether, ester, or amide linkage with the group Z; and

when Z is hydroxyl, formula V includes a luciferin dimer connected atthe two A rings via an —OCH₂O— bridge; and

wherein

A′ and B′ are optional aromatic rings fused to ring A, only one of whichmay be present at a time, so as to form a fused tricyclic system; and

when B′ is present, the group Z is present, and

when A′ is present, the group Z is absent; and

wherein

one carbon of ring A may be replaced by an N-oxide moiety;

the dotted line in ring B is an optional double bond;

if X is N═C, ring C is attached at the carbon atom of the N═C moiety;and

A⁻ is an anion, present when a quaternary nitrogen is present; or a saltthereof, with the proviso that W is not hydrogen when the compound towhich W is attached is luciferin, luciferin methyl ester, oraminoluciferin or when rings A and B form a naphthalene or quinolinering system.

Further derivatives of luciferin or aminoluciferin have the generalformula VI:

whereinR¹ represents hydrogen, hydroxyl, amino, C₁₋₂₀alkoxy, substitutedC₁₋₂₀alkoxy, C₂₋₂₀alkenyloxy, substituted C₂₋₂₀alkenyloxy, halogenatedC₁₋₂₀alkoxy, substituted halogenated C₁₋₂₀alkoxy, C₃₋₂₀alkynyloxy,substituted C₃₋₂₀alkynyloxy, C₃₋₂₀cycloalkoxy, substitutedC₃₋₂₀cycloalkoxy, C₃₋₂₀cycloalkylamino, substitutedC₃₋₂₀cycloalkylamino, C₁₋₂₀alkylamino, substituted C₁₋₂₀alkylamino,diC₁₋₂₀alkylamino, substituted diC₁₋₂₀alkylamino, C₂₋₂₀alkenylamino,substituted C₂₋₂₀alkenylamino, diC₂₋₂₀alkenylamino, substituteddiC₂₋₂₀alkenylamino, C₂₋₂₀alkenylC₁₋₂₀alkylamino, substitutedC₂₋₂₀alkenylC₁₋₂₀ alkylamino, C₃₋₂₀alkynylamino, substitutedC₃₋₂₀alkynylamino, diC₃₋₂₀alkynylamino, substituted diC₃₋₂₀alkynylamino,C₃₋₂₀alkynylC₂₋₂₀alkenylamino,or substituted C₃₋₂₀alkynylC₂₋₂₀alkenylamino;R⁶ represents CH₂OH; COR¹¹ wherein R¹¹ represents H, OH, C₁₋₂₀alkoxide,C₂₋₂₀ alkenyl, or NR¹²R¹³ wherein R¹² and R¹³ are independently H orC₁₋₂₀alkyl; or —OM⁺ wherein M⁺ is an alkali metal; or a salt; andR⁷ represents H, C₁₋₆alkyl, C₂₋₂₀alkenyl, halogen, or C₁₋₆alkoxide,

with the proviso that when R¹ is OH or NH₂, R⁷ is not H, R⁶ is not COR¹¹wherein R¹¹ is OH or OMe formula VI does not include luciferin,luciferin methyl ester, and aminoluciferin.

Further derivatives of luciferin or aminoluciferin have the generalformula VII:

whereinY is N-oxide, N-loweralkyl, or CH;X is S or CH═CH; orY is N and X is N═C or CH═CH;Z and Z′ are H, OR, NHR, or NRR; orZ is a cyclic dietherified dihydroxyborane group attached to ring A viathe boron atom;

Z″ is O, S, NH, NHR, or N═N;

each W is independently H, halo, C₁₋₆alkyl, C₂₋₂₀alkenyl, hydroxyl, orC₁₋₆alkoxy;

each of K¹, K², K³, and K⁴ are independently CH, N,N-oxide, orN-loweralkyl;

R is H, C₁₋₂₀alkyl, substituted C₁₋₂₀alkyl, C₂₋₂₀alkenyl, substitutedC₂₋₂₀alkenyl, halogenated C₂₋₂₀alkenyl, substituted halogenatedC₂₋₂₀alkenyl, C₂₋₂₀alkenylC₁₋₂₀alkyl, substitutedC₂₋₂₀alkenylC₁₋₂₀alkyl, C₃₋₂₀alkynyl, substituted C₃₋₂₀alkynyl,C₃₋₂₀alkynylC₂₋₂₀alkenyl, substituted C₃₋₂₀alkynylC₂₋₂₀alkenyl,C₃₋₂₀cycloalkyl, substituted C₃₋₂₀cycloalkyl, C₆₋₃₀aryl, heteroaryl,C₆₋₃₀arylC₁₋₂₀alkyl, substituted C₆₋₃₀aryl, substituted heteroaryl,substituted C₆₋₃₀arylC₁₋₂₀alkyl, C₁₋₂₀alkoxycarbonyl,C₆₋₃₀arylC₁₋₂₀alkoxycarbonyl, C₆₋₃₀arylthioC₁₋₂₀alkyl,hydroxyC₁₋₂₀alkyl, triC₁₋₂₀ammoniumC₁₋₂₀alkyl, heteroarylC₁₋₂₀alkyl,substituted heteroarylC₁₋₂₀alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen,N-methyl-tetrahydropyridinyl, pentafluorophenylsulphonyl, and M⁺ when Z″is oxygen, wherein M is an alkali metal; wherein the alkyl, cycloalkyl,alkenyl, and/or alkynyl groups may be optionally substituted by one moreC₁₋₂₀alkyl, halo, hydroxyl, acetyl, amino, lower alkylamino, loweralkynylamino, imidazolinylmethylamino, di-lower alkylamino, di-loweralkynylamino, piperidino, pyrrolidino, azetidino, aziridino,di-imidazolinylmethylamino, mercapto, C₁₋₂₀alkylthio,

C₆₋₃₀arylthio, or trifluoromethyl groups; and each group R is definedindependently if more than one is present;

Q is (C═O)_(n) or (CH₂)_(n);

n is 0, 1, or 2;

and wherein

when Z is amino, one or both of the hydrogens may be replaced byC₁₋₂₀alkyl, or the group L, wherein

L is an amino acid radical, a peptide radical having up to 20 amino acidmoieties, or may be any other small molecule that is a substrate for anonluciferase;

and wherein

when Z is hydroxyl or amino, H may be replaced by (HO)₂P(O)—OCH₂—,sulfo, or

—PO₃H₂, or by a cephalosporanic acid attached to the group Z via acarbon chain of one or more carbon atoms; and

when Z is hydroxyl or amino or when Z″—R is hydroxyl, H may be replacedby the group L′-linker, wherein L′ is a group removable by an enzyme tofree the linker, and linker is carbon chain that can self-cleave,optionally interrupted by one or more nitrogen atoms, oxygen atoms,carbonyl groups, (substituted)aromatic rings, or peptide bonds, and

linker is attached to L′ via an oxygen atom or an NH group at oneterminus of the linker and the other terminus of the linker forms anether, ester, or amide linkage with the group Z; and

when Z is hydroxyl, formula VII includes a luciferin dimer corrected atthe two A rings via an —OCH₂O— bridge; and

wherein

A′ and B′ are optional aromatic rings fused to ring A, only one of whichmay be present at a time, so as to form a fused tricyclic system; and

when B′ is present, the group Z is present, and

when A′ is present, the group Z is absent; and

wherein

one carbon of ring A may be replaced by an N-oxide moiety;

the dotted line in ring B is an optional double bond;

if X is N═C, ring C can optionally be attached at the carbon atom of theN═C moiety; and

A⁻ is an anion, present when a quaternary nitrogen is present; or a saltthereof; with the proviso that W is not hydrogen when rings A and B forma naphthalene ring system.

-   -   Other derivatives include a compound of formula VIII:

wherein

n=0 or 1 and when

n=0, then X═S, and

is a single bond; or when

n=1, then X═CH, and

is a double bond;

R₁═H, F, or OH;

R₂═H, methyl, ethyl, propyl, butyl, benzyl, hydroxyethyl, or an ester ofhydroxyethyl;

R₃ and R₄ are independently H, methyl, ethyl, propyl, allyl,imidazolinylmethyl, or

R₃ and R₄ together with the nitrogen atom to which they are attachedform a piperidino, pyrrolidino, azetidino, or aziridino ring;

R₅ and R₆ are independently H or methyl;

R₇ is H or methyl;

R₈ is H, methyl, hydroxyl, or acetyl; and

R₉ is H or methyl.

Compounds of formula VIII may be useful as MAO substrates.

Yet other derivatives include a compound of formula IX:

wherein R¹ is H, OR, NH—C(O)—O-benzyl, or NH—O-iso-butyl;

R is lower alkyl, benzyl, 2,4,6-trimethylbenzyl, phenylpiperazinobenzyl,o-trifluoromethylbenzyl, or 3-picolinyl;

R⁶ is a carboxyl group esterified by lower alkyl, 3-picolinyl, ethyleneglycol when R¹ is H or OR, or R⁶ is carboxyl when R¹ is NH—C(O)—O-benzylor NH—O-iso-butyl or when R¹ is OR wherein R is 2,4,6-trimethylbenzyl,phenylpiperazinobenzyl, o-trifluoromethylbenzyl. Such derivatives may beuseful as P450 substrates.

Also provided is a compound of formula X:

wherein

Y is N,N-oxide, N-loweralkyl, or CH;

X is S, CH═CH, or N═C,

Z and Z′ are independently H, OR, NHR, or NRR;

Z″ is O, S, NH, NHR, or N═N;

each W is independently H, halo, C₁₋₆alkyl, C₂₋₂₀alkenyl, hydroxyl, orC₁₋₆alkoxy; or

W and Z are both keto groups on ring A, and the dotted lines in ring Aare absent;

each of K¹, K², K³, and K⁴ are independently CH, N,N-oxide, orN-loweralkyl, and the dotted lines between K¹ and K², and K³ and K⁴,denote optional double bonds;

R is H, amino, C₁₋₂₀alkyl, C₂₋₂₀alkenyl, halogenated C₂₋₂₀alkenyl,C₃₋₂₀alkynyl, C₂₋₂₀alkenylC₁₋₂₀alkyl, C₃₋₂₀alkynylC₂₋₂₀alkenyl,C₃₋₂₀cycloalkyl, C₆₋₃₀aryl, heteroaryl, C₆₋₃₀arylC₁₋₂₀alkyl,C₁₋₁₂alkoxy, C₁₋₂₀alkylsulfoxy, C₆₋₃₀arylsulfoxy,C₆₋₃₀arylsulfoxyC₁₋₂₀alkyl, C₁₋₂₀alkylsulfoxyC₁₋₂₀alkyl,C₁₋₂₀alkoxycarbonyl, C₆₋₃₀arylC₁₋₂₀alkoxycarbonyl,C₆₋₃₀arylthioC₁₋₂₀alkyl, hydroxyC₁₋₂₀alkyl, triC₁₋₂₀ammoniumC₁₋₂₀alkyl,heteroarylsulfoxy, heteroarylC₁₋₂₀alkyl, heteroaryl having quaternarynitrogen, heteroarylcarbonyl having quaternary nitrogen,N-methyl-tetrahydropyridinyl; or M⁺ when Z″ is oxygen, wherein M is analkali metal;

wherein any alkyl, alkenyl, alkynyl, cycloalkyl, alkoxy, aryl, orheteroaryl groups of R can be optionally substituted by one or more,e.g., 1, 2, 3, 4, or 5, C₁₋₂₀alkyl, halo, hydroxyl, acetyl, —COOR¹,—SO₃R¹, amino, nitro, lower alkylamino, lower alkynylamino,imidazolinylmethylamino, di-lower alkylamino, di-lower alkynylamino,piperidino, pyrrolidino, azetidino, aziridino,di-imidazolinylmethyl-amino, mercapto, C₁₋₂₀alkylthio, C₆₋₃₀arylthio,trifluoromethyl, C₁₋₂₀alkylcarboxyl, C₆₋₃₀aryl, substituted C₆₋₃₀aryl,C₆₋₃₀arylC₁₋₂₀alkoxyl, heterocycle C₁₋₂₀alkyl, substitutedC₆₋₃₀arylC₁₋₂₀alkoxyl, C₆₋₃₀arylC₁₋₂₀alkyl carbonyl, substitutedC₆₋₃₀arylC₁₋₂₀alkyl carbonyl or additional unsubstituted R groups; andwherein each group R is defined independently if more than one ispresent;

wherein heterocycle C₁₋₂₀alkyl is optionally substituted with one ormore, e.g., 1,2,3,4, or 5, R groups;

R¹ is hydrogen or C₁₋₆alkyl;

Q is C(═O) or CH₂;

n is 0, 1, or 2;

and wherein

when Z or Z″ is amino, one or both of the hydrogens of the amino groupmay be replaced by C₁₋₂₀alkyl, or the group L, wherein

L is an amino acid radical, a peptide radical having up to 20 amino acidmoieties, or any other small molecule that is a substrate for anonluciferase; with the proviso that when L is an amino acid radical ora peptide radical, W is not H;

and wherein

when Z is hydroxyl or amino, H of the hydroxyl or amino may be replacedby (HO)₂P(O)—OCH₂—, sulfo,

—PO₃H₂, or by a cephalosporanic acid attached to the group Z via acarbon chain of one or more carbon atoms; with the proviso that whenring B is a thiazole ring, the sulfo or the —PO₃H₂ group is attached tothe hydroxyl oxygen via a loweralkylene chain; and

when Z or Z′ is hydroxyl or amino or when Z″—R is hydroxyl, one H of thehydroxyl or amino may be replaced by the group L′-linker, wherein L′ isa group removable by an enzyme to free the linker, and linker is carbonchain that can self-cleave, optionally interrupted by one or morenitrogen atoms, oxygen atoms, carbonyl groups, (substituted)aromaticrings, or peptide bonds, and

linker is attached to L′ via an oxygen atom or an NH group at oneterminus of the linker and the other terminus of the linker forms anether, ester, or amide linkage with the group Z, Z′, or Z″—R; and

when Z is OR, formula X can optionally be a dimer connected at the two Arings via a CH₂ or CH₂—C₆H₄—CH₂ bridge, and the R group of each Z groupconnecting the dimer of formula X is replaced by the bridge; and

wherein

A′ and B′ are optional aromatic rings fused to ring A, only one of whichmay be present at a time, so as to form a fused tricyclic system; and

when B′ is present, the group Z is present, and

when A′ is present, the group Z is absent; and

wherein

one carbon of ring A may be replaced by an N-oxide moiety;

the dotted line in ring B is an optional double bond;

if X is N═C, ring C can optionally be attached at the carbon atom of theN═C moiety; and

A⁻ is an anion, present when a quaternary nitrogen is present; or a saltthereof, with the proviso that W is not hydrogen when the compound towhich W is attached is luciferin, luciferin methyl ester, oraminoluciferin or when rings A and B form a naphthalene or quinolinering system,

with the proviso that -Q-Z″—R is not —C(O)—NH—NH₂ when a ring Asubstituent is OH.

In one embodiment, the W group attached to ring C is absent (i.e., thevalue of “n” is 0). In another embodiment, the W group attached to ringC is H or F.

Further provided is a compound of formula XI:

wherein

Y is N-oxide, N-loweralkyl, or CH;

X is S or CH═CH; or

Y is N and X is N═C or CH═CH;

Z and Z′ are H, OR, NHR, or NRR; or

Z is a cyclic dietherified dihydroxyborane group attached to ring A viathe boron atom;

Z″ is O, S, NH, NHR, or N═N;

each W is independently H, halo, C₁₋₆alkyl, C₂₋₂₀alkenyl, hydroxyl, orC₁₋₆alkoxy;

each of K¹, K², K³, and K⁴ are independently CH, N,N-oxide, orN-loweralkyl, and the dotted lines between K¹ and K², and K³ and K⁴,denote optional double bonds;

R is H, C₁₋₂₀alkyl, C₂₋₂₀alkenyl, halogenated C₂₋₂₀alkenyl,C₃₋₂₀alkynyl, C₂₋₂₀alkenylC₁₋₂₀alkyl, C₃₋₂₀alkynylC₂₋₂₀alkenyl,C₃₋₂₀cycloalkyl, C₆₋₃₀aryl, heteroaryl, C₆₋₃₀arylC₁₋₂₀alkyl,C₁₋₂₀alkylsulfoxy, C₆₋₃₀arylsulfoxy, C₆₋₃₀arylsulfoxyC₁₋₂₀alkyl,C₁₋₂₀alkylsulfoxyC₁₋₂₀alkyl, C₁₋₂₀alkoxycarbonyl,C₆₋₃₀arylC₁₋₂₀alkoxycarbonyl, C₆₋₃₀arylthioC₁₋₂₀alkyl,hydroxyC₁₋₂₀alkyl, triC₁₋₂₀ammoniumC₁₋₂₀alkyl, heteroaryl-sulfoxy,heteroarylC₁₋₂₀alkyl, heteroaryl having quaternary nitrogen,heteroarylcarbonyl having quaternary nitrogen,N-methyl-tetrahydropyridinyl; or M⁺ when Z″ is oxygen, wherein M is analkali metal;

wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroarylgroups of R can be optionally substituted by one or more, e.g., 1, 2, 3,4, or 5, C₁₋₂₀alkyl, halo, hydroxyl, acetyl, —COOR¹, —SO₃R¹, amino,nitro, lower alkylamino, lower alkynylamino, imidazolinylmethylamino,di-lower alkylamino, di-lower alkynylamino, piperidino, pyrrolidino,azetidino, aziridino, di-imidazolinylmethyl-amino, mercapto,C₁₋₂₀alkylthio, C₆₋₃₀arylthio, trifluoromethyl, substituted C₆₋₃₀aryl,C₁₋₂₀alkylcarboxyl, substituted C₆₋₃₀aryl, substitutedC₆₋₃₀arylC₁₋₂₀alkoxyl, substituted C₆₋₃₀arylC₁₋₂₀alkyl carbonyl oradditional unsubstituted R group; and each group R is definedindependently if more than one is present;

R¹ is hydrogen or C₁₋₆alkyl;

Q is C(═O) or CH₂;

each n is independently 0, 1, or 2;

and wherein

when Z is amino, one or both of the hydrogens of the amino group may bereplaced by C₁₋₂₀alkyl, or the group L, wherein

L is an amino acid radical, a peptide radical having up to 20 amino acidmoieties, or may be any other small molecule that is a substrate for anonluciferase;

and wherein

when Z or Z′ is hydroxyl or amino, H of the hydroxyl or amino may bereplaced by (HO)₂P(O)—OCH₂—, sulfo, —PO₃H₂, or by a cephalosporanic acidattached to the group Z via a carbon chain of one or more carbon atoms;and

when Z is hydroxyl or amino or when Z″—R is hydroxyl, one H of thehydroxyl or amino may be replaced by the group L′-linker, wherein L′ isa group removable by an enzyme to free the linker, and linker is carbonchain that can self-cleave, optionally interrupted by one or morenitrogen atoms, oxygen atoms, carbonyl groups, (substituted)aromaticrings, or peptide bonds, and

linker is attached to L′ via an oxygen atom or an NH group at oneterminus of the linker and the other terminus of the linker forms anether, ester, or amide linkage with the group Z, Z′, or Z″—R; and

when Z is hydroxyl, formula XI includes a luciferin dimer connected atthe two A rings via an —OCH₂O— bridge; and

wherein

A′ and B′ are optional aromatic rings fused to ring A, only one of whichmay be present at a time, so as to form a fused tricyclic system; and

when B′ is present, the group Z is present, and

when A′ is present, the group Z is absent; and

wherein

the dotted line in ring B is an optional double bond;

if X is N═C, ring C can optionally be attached at the carbon atom of theN═C moiety; and

A⁻ is an anion, present when a quaternary nitrogen is present; or a saltthereof; with the proviso that W is not hydrogen when rings A and B forma naphthalene ring system. In one embodiment, the W group attached toring C is absent (i.e., the value of “n” is 0). In another embodiment,the W group attached to ring C is H or F.

Also provided is a compound of formula XII:

wherein

Y is N,N-oxide, N-loweralkyl, or CH;

X is S, CH═CH, or N═C,

Z and Z′ are independently H, OR, NHR, or NRR;

Z″ is O, S, NH, NHR, or N═N;

each W is independently H, halo, C₁₋₆alkyl, C₂₋₂₀alkenyl, hydroxyl, orC₁₋₆alkoxy; or

W and Z are both keto groups on ring A, and the dotted lines in ring Aare absent;

each of K¹, K², K³, and K⁴ are independently CH, N,N-oxide, orN-loweralkyl, and the dotted lines between K¹ and K², and K³ and K⁴,denote optional double bonds;

R is H, C₁₋₂₀alkyl, C₂₋₂₀alkenyl, halogenated C₂₋₂₀alkenyl,C₃₋₂₀alkynyl, C₂₋₂₀alkenylC₁₋₂₀alkyl, C₃₋₂₀alkynylC₂₋₂₀alkenyl,C₃₋₂₀cycloalkyl, C₆₋₃₀aryl, heteroaryl, heterocyclic, substitutedheterocyclic, C₆₋₃₀arylC₁₋₂₀alkyl, C₁₋₂₀alkylsulfoxy, C₆₋₃₀arylsulfoxy,C₆₋₃₀arylsulfoxyC₁₋₂₀alkyl, C₁₋₂₀alkylsulfoxyC₁₋₂₀alkyl,C₁₋₂₀alkoxycarbonyl, C₆₋₃₀arylC₁₋₂₀alkoxycarbonyl,C₆₋₃₀arylthioC₁₋₂₀alkyl, hydroxyC₁₋₂₀alkyl, triC₁₋₂₀ammoniumC₁₋₂₀alkyl,heteroarylsulfoxy, heteroarylC₁₋₂₀alkyl, heteroaryl having quaternarynitrogen, heteroarylcarbonyl having quaternary nitrogen,N-methyl-tetrahydropyridinyl; or M⁺ when Z″ is oxygen, wherein M is analkali metal;

wherein the alkyl, alkenyl, alkynyl, cycloalkyl, aryl, and heteroarylgroups of R can be optionally substituted by one or more, e.g., 1, 2, 3,4, or 5, C₁₋₂₀alkyl, halo, hydroxyl, acetyl, —COOR¹, —SO₃R¹, amino,nitro, lower alkylamino, lower alkynylamino, imidazolinylmethylamino,di-lower alkylamino, di-lower alkynylamino, piperidino, pyrrolidino,azetidino, aziridino, di-imidazolinylmethyl-amino, mercapto,C₁₋₂₀alkylthio, C₆₋₃₀arylthio, trifluoromethyl, C₁₋₂₀alkylcarboxyl,C₆₋₃₀aryl, substituted C₆₋₃₀aryl, C₆₋₃₀arylC₁₋₂₀alkoxyl, substitutedC₆₋₃₀arylC₁₋₂₀alkoxyl, C₆₋₃₀arylC₁₋₂₀alkyl carbonyl, substitutedC₆₋₃₀arylC₁₋₂₀alkyl carbonyl or additional unsubstituted R groups; andwherein each group R is defined independently if more than one ispresent;

wherein substituted aryl groups are substituted with one or alkyl,alkenyl, alkynyl, cycloalkyl, aryl, heterocyclic, substitutedheterocyclic, and heteroaryl groups of R can be optionally substitutedby one or more C₁₋₂₀alkyl, C₆-C₁₀aryl, halo, hydroxyl, acetyl, —COOR¹,amino, nitro, lower alkylamino, lower alkynylamino,imidazolinylmethylamino, di-lower alkylamino, di-lower alkynylamino,piperidino, pyrrolidino, azetidino, aziridino,di-imidazolinylmethylamino, mercapto, C₁₋₂₀alkylthio, C₆₋₃₀arylthio, ortrifluoromethyl groups;

R¹ is hydrogen or C₁₋₆alkyl;

Q is C(═O) or CH₂;

n is 0, 1, or 2;

and wherein

when Z or Z″ is amino, one or both of the hydrogens of the amino groupmay be replaced by C₁₋₂₀alkyl, or the group L, wherein

L is an amino acid radical, a peptide radical having up to 20 amino acidmoieties, or any other small molecule that is a substrate for anonluciferase; with the proviso that when L is an amino acid radical ora peptide radical, W is not H;

and wherein

when Z is hydroxyl or amino, H may be replaced by (HO)₂P(O)—OCH₂—,sulfo, or —PO₃H₂, or by a cephalosporanic acid attached to the group Zvia a carbon chain of one or more carbon atoms; with the proviso thatwhen ring B is a thiazole ring, the sulfo or the —PO₃H₂ group isattached to the hydroxyl oxygen via a loweralkylene chain; and

when Z or Z′ is hydroxyl or amino or when Z″—R is hydroxyl, one H may bereplaced by the group L′-linker, wherein L′ is a group removable by anenzyme to free the linker, and linker is carbon chain that canself-cleave, optionally interrupted by one or more nitrogen atoms,oxygen atoms, carbonyl groups, (substituted)aromatic rings, or peptidebonds, and

linker is attached to L′ via an oxygen atom or an NH group at oneterminus of the linker and the other terminus of the linker forms anether, ester, or amide linkage with the group Z, Z′, or Z″—R; and

when Z is hydroxyl, formula XII includes a luciferin dimer connected atthe two A rings via an —OCH₂O— bridge; and

wherein

A′ and B′ are optional aromatic rings fused to ring A, only one of whichmay be present at a time, so as to form a fused tricyclic system; and

when B′ is present, the group Z is present, and

when A′ is present, the group Z is absent; and

wherein

the dotted line in ring B is an optional double bond; if X is N═C, ringC can optionally be attached at the carbon atom of the N═C moiety; and

A⁻ is an anion, present when a quaternary nitrogen is present; or a saltthereof, with the proviso that W is not hydrogen when the compound towhich W is attached is luciferin, luciferin methyl ester, oraminoluciferin or when rings A and B form a naphthalene or quinolinering system.

In one embodiment, the W group attached to ring C is absent (i.e., thevalue of “n” is 0). In another embodiment, the W group attached to ringC is H or F.

IV. Exemplary Luciferin Derivatives

Derivatives having a six membered B ring include:

wherein L may be any structure for an enzyme, such as a peptide sequenceor small molecules; X is O, NH, or a self-cleavable linker; Z is N(aminoquinolinyl luciferin) or CH (naphthyl luciferin); Y is O (ester),NH (amide), NH—NH (hydrazide), or S (thioester); and R may be alkyl,aromatic, a peptide, an oligonucleotide, or a self-cleavable linkerattached to a substrate for an enzyme.

In one embodiment, a derivative may be employed to detectN-dealkylation, e.g., by cytochrome (CYP) P450 isozymes. In the case ofan ester (R=alkyl chain), an esterase may be added after thedealkylation reaction is commenced, or at the time the dealkylationreaction is initiated, and before a luciferase-mediated reaction isinitiated.

Although light output with 6-aminoquinolinyl luciferin (formula V) is pHsensitive, reactions including control reactions may be conducted at thesame pH. Alternatively, 6-aminoquinolinyl luciferin may be modified soas to alter electron density distribution of the aromatic ring, forexample, with halogenation.

Derivatives of the invention include fluorinated luciferin, quinolinylluciferin, aminoquinolinyl luciferin, and naphthyl luciferin. Forinstance, the optical properties of fluorinated derivatives ofaminoquinolinyl luciferin may be altered due to the electron withdrawingpower of fluorine. Fluorinated derivatives include a compound of formulaXXII and XXIII:

wherein X═O or NH; Y═N or CH; and F may be at 3′, 4′, 5′, 7′, or 8′

wherein X═O or NH; and F may be at 4′, 5′, or 7′.

An exemplary fluorinated derivative is 5-fluoro-6-aminoluciferin (FIG.3).

In one embodiment, the invention provides for quinolinyl derivatives(compounds of formula XXIV-XXVI):

Derivatives of the invention may be employed as sensors for reactiveoxygen species (ROS) or a redox sensor for horseradish peroxidase (HRP)and other enzymes. Those derivatives include compounds of formulaXXVII-XXXII:

Exemplary bioluminogenic MAO substrates include:

Exemplary bioluminogenic FMO substrates include formula XXXIV

The A ring of D-luciferin may also be modified to include another ringstructure (compound of formulas XXXV-XXXVI):

Other A ring modifications include a stable substrate for phosphatasewhich may also be a luciferase substrate (compound of formula XXXVII):

The C ring of luciferin may be modified to include a substrate foracetylcholinesterase (ACh), which optionally has a modified A ring or amodified C and A ring. ACh has a very high turnover number (25,000 s⁻¹)and has attained kinetic perfection as indicated by its k_(cat)/K_(M)value of 2×10⁸ M⁻¹S⁻¹. Two types of derivatives which are substrates forACh include (compounds of formula XXXIX-XXXX):

Derivatives useful to detect β-lactamase-mediated reactions includequinolinyl luciferin, aminoquinolinyl luciferin and naphthyl luciferinderivatives, optionally also modified with a halogen, e.g., a A ringmodification that replaces a H linked to a C ring atom with a F.

wherein R=different group, X═N or CH, Y═S or CH═CH, and Z═O or NH

Derivatives which include esters of luciferin (a C ring modification)optionally with a A ring modification or a B ring modification, or a Aand a C ring modification, include compound of formulas XXXXIV-LVIII:

To prepare esters for a derivative of luciferin that are substrates forenzymes including P450, MAO and GST, a cyclization procedure, may beemployed with TCEP to reduce ethylene glycol ester D-cysteine beforeadding to a cyano-containing molecule, is utilized. For instance, to asolution of D-cysteine ester neutralized with potassium bicarbonate, addTECP. After a few minutes, add the solution to the appropriate nitriledissolved in methanol. Also, methyl ester luciferin derivatives are madeby direct cyclization of benzothiole derivatives with methyl esterD-cysteine or methylation of carboxylic acids of luciferin withdiazomethane.

Derivatives of luciferin with A, B and/or C ring modifications include:

-   -   (compounds of formula LXI-LXX below)

Yet further derivatives include compounds of formula LXXI, LXXII andLXXIII (below)

wherein R₁═H, F, or OH;wherein R₅ and R₆ are independently H or CH₃.

In one embodiment, the derivative is a GST substrate (formulasLXXIV-LXXV)

Exemplary phosphatase and sulfatase substrates include formulasLXXX-LXXXIII:

other derivatives of the invention include compounds of formulaLXXXVIII-LXXXIX:

Derivatives for use in redox or dealkylase bioluminescent assays includecompounds of formulas LXXXX-LXXXXI:

Specific derivatives useful as reduction sensors or a dealkylase sensorinclude compounds of formula LXXXXII-LXXXXVI:

Other examples of redox sensors are shown in FIGS. 4 and 6.Other esterase substrates are shown in FIGS. 8-10, 22-23, 26, 37B, 40and 42.

V. Derivatives of Fluorophores

In one embodiment, derivatives of a fluorophore of the invention havethe following structure L-F, where F is a fluorophore and L is asubstrate linked via O to the fluorophore. In one embodiment,fluorescent substrates according to the present invention are benzopyranderivatives having the general formula XIII:

whereinthe dashed ring is an optionally present benzo ring;the dashed bond in the B ring is present only when the dashed ring isabsent;X′ is CH when the dashed ring is absent;X′ is NH when the dashed ring is present;X′ is a carbon atom when the dashed ring is present and X′ forms part ofa spiro ring system which is a γ-butyrolactone ring having an optionallysubstituted benzo ring fused at the α and β carbons of the lactone ringand attached to X′ at the γ carbon;W¹, W³, and W⁴ are independently H, halo, carboxyl, carboxy ester,loweralkyl, hydroxyloweralkyl, C₆₋₂₀aryl, or substituted C₆₋₂₀aryl; W²is hydroxyl, loweralkoxy, or amino, wherein one or both amino hydrogensmay be replaced by lower alkyl;Z¹ is a loweralkylene chain terminated by an amino group, aloweralkylamino group, a diloweralkylamino group, a thiol group, or alower alkylthio group; and W² is alkoxy, or OCH(R₇)CH(R₈)CH(R₉)N(R₃R₄);andZ² is a keto group present only when the benzo ring is absent.

Exemplary fluorogenic MAO substrates include formulas LXXXXVIII-C:

Fluorogenic FMO substrates include formulas CI-CIII:

See FIGS. 5 and 11 for exemplary fluorogenic substrates for redox andMAO reactions.

Any fluorophore may be employed to prepare a fluorogenic substrate,fluorophores including but not limited to fluoroscein, Texas Red, DAPI,PI, acridine orange, Alexa fluors, e.g., Alexa 350, Alexa 405 or Alexa488, cyanine dyes, e.g., Cy3, coumarin, ethidium bromide, fluorescein,BODIPY, a rhodol, Rox, 5-carboxyfluorescein, 6-carboxyfluorescein, ananthracene, 2-amino-4-methoxynapthalene, a phenalenone, an acridone,fluorinated xanthene derivatives, α-naphtol, β-napthol, 1-hydroxypyrene,coumarins, e.g., 7-amino-4-methylcoumarin (AMC) or7-amino-4-trifluoromethylcoumarin (AFC), rhodamines, e.g.,tetramethylrhodamine, rhodamine-110, or carboxyrhodamine, cresyl violet,or resorufin, as well as fluorophores disclosed in U.S. Pat. No.6,420,130, the disclosure of which is incorporated by reference herein.

VI. Linkers

A linker strategy may be employed for either the A or C ring modifiedluciferins, or with fluorophore containing derivatives, to introduce asubstrate for an enzyme of interest such as a deacetylase, deformylase,demethylase or other enzyme that can remove the L group of formula I (asubstrate for that enzyme) to free the linker, yielding a substrate ofluciferase, or a prosubstrate, where the remaining linker may optionallybe removed by a nonenzymatic reaction.

Linkers can be alkyl or alkoxy chains, such as (C₁-C₆)alkyl or(C₁-C₆)alkoxy groups. The chain can have one or more electronwithdrawing group substituents R, such as an aldehyde, acetyl,sulfoxide, sulfone, nitro, cyano group, or a combination thereof. Otherlinkers include trimethyl lock, quinine methide and diketopiperazinelinkers, and their derivatives. A trimethyl lock linker can beillustrated as follows:

wherein R is as defined for any one of formulas I-III or V-XII, which isa group removable by an enzyme, e.g., an enzyme that is being assayed;the trimethyl lock linker replaces a hydrogen atom of one of the groupsZ, Z′, or Z″—R; and ‘leaving group’ is the remainder of the structure offormula I-III or V-XII. See Wang et al., J. Org. Chem., 62:1363 (1997)and Chandran et al., J. Am. Chem. Soc., 127:1652 (2005) for the use oftrimethyl lock linkers.

A quinine methide linker can be illustrated as follows:

wherein R is as defined for any one of formulas I-III or V-XII, which isa group removable by an enzyme, e.g., an enzyme that is being assayed;the quinine methide linker replaces a hydrogen atom of one of the groupsZ, Z′, or Z″—R; and ‘leaving group’ is the remainder of the structure offormula I-III or V-XII. See Greenwald et al., J. Med. Chem., 42:3657(1999) and Greenwald et al., Bioconjugate Chem., 14:395 (2003) for theuse of quinine methide linkers.A diketopiperazine linker can be illustrated as follows:

wherein R is as defined for any one of formulas I-III or V-XII, which isa group removable by an enzyme, e.g., an enzyme that is being assayed;each R′ of the diketopiperazine linker is independently H or an alkylchain optionally interrupted by O, S, or NH, preferably a methyl group;the diketopiperazine linker replaces a hydrogen atom of one of thegroups Z, Z′, or Z″—R; and ‘leaving group’ is the remainder of thestructure of formula I-III or V-XII. See Wei et al., Bioorg. Med. Chem.Lett., 10: 1073 (2000) for the use of diketopiperazine linkers.Other linker containing derivatives include:

β-elimination of a product of a bioluminogenic or fluorogenic reactionwith a substrate having a group R that in the product is an electronwithdrawing group R, such as aldehyde, acetyl, sulfoxide, sulfone,nitro, or cyano, may yield a substrate for luciferase or a fluorophore.

Any fluorogenic chromophore or luminophore

For instance, to detect MAO, the derivative may undergo the followingreaction.

Any fluorogenic chromophore or luminophoreTo detect FMO, the derivative may undergo the following reaction:

Any fluorogenic chromophore or luminophore

Specific embodiments of reactions and derivatives having formulasCXI-CXV which employ a linker include:

wherein L=Me for demethylase, Ac for deacetylase, or CHO fordeformylase, X═S or CH═CH, Y═N or CH, and Z═O or NH;

wherein R=Me for demethylase, Ac for deacetylase, or CHO fordeformylase, X═S or CH═CH, Y═N or CH, and Z═O or NH

wherein L=Me for demethylase, Ac for deacetylase, or CHO fordeformylase, X═S or CH═CH, and Y═N or CH;

wherein L=Me for demethylase, Ac for deacetylase, or CHO fordeformylase, X═S or CH═CH, Y═N or CH, and Z═O or NH; or

wherein L=Me for demethylase, Ac for deacetylase, or CHO fordeformylase, X═S or CH═CH, Y═N or CH, and Z═O or NH.

VII. Agents Useful to Stabilize Light Production in Luciferase-MediatedReactions

Agents useful to stabilize light production in a luciferase-mediatedreaction include organic compounds (i.e., compounds that comprise one ormore carbon atoms). Such an agent may be added prior to, at theinitiation of and/or during a nonluciferase enzyme-mediated reaction ora luciferase-mediated reaction. Suitable organic compounds can comprisea carbon-sulfur bond or a carbon-selenium bond, for example suitableorganic compounds can comprise a carbon-sulfur double bond (C═S), acarbon selenium double bond (C═Se), a carbon-sulfur single bond (C—S),or carbon-selenium single bond (C—Se). Suitable organic compounds canalso comprise a carbon bound mercapto group (C—SH) or a sulfur atombound to two carbon atoms (C—S—C). In one embodiment, compounds arelipophilic in nature.

Suitable compounds that comprise a carbon sulfur double bond or a carbonselenium double bond include for example compounds of formula (XIV):

wherein X is S or Se; R₁ and R₂ are each independently hydrogen,(C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₂₀)alkoxy, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, aryl, heteroaryl, or NR_(a)R_(b); or R₁ and R₂ togetherwith the carbon to which they are attached form a 5, 6, 7, or 8 memberedsaturated or unsaturated ring comprising carbon and optionallycomprising 1, 2, or 3 heteroatoms selected from oxy (—O—), thio (—S—),or nitrogen (—NR_(c))—, wherein said ring is optionally substituted with1, 2, or 3 halo, hydroxy, oxo, thioxo, carboxy, (C₁-C₂₀)alkyl,(C₃-C₈)cycloalkyl, (C₁-C₂₀)alkoxy, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, aryl, orheteroaryl; and R_(a), R_(b) and R_(c) are each independently hydrogen,(C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl, (C₂-C₂₀)alkynyl, aryl, heteroaryl; wherein any(C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₂₀)alkoxy, (C₂-C₂₀)alkenyl(C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkoxycarbonyl, or (C₂-C₂₀)alkynyl of R₁, R₂,R_(a), R_(b), and R_(c) is optionally substituted with one or more (e.g1, 2, 3, or 4) halo, hydroxy, mercapto, oxo, thioxo, carboxy,(C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkoxycarbonyl, aryl, or heteroaryl; andwherein any aryl or heteroaryl is optionally substituted with one ormore (1, 2, 3, or 4) halo, hydroxy, mercapto, carboxy, cyano, nitro,trifluoromethyl, trifluoromethoxy, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkanoyloxy, sulfo or (C₁-C₂₀)alkoxycarbonyl; or a salt thereof.

Suitable compounds that comprise a mercapto group include for examplecompounds of the formula R₃SH wherein: R₃ is (C₁-C₂₀)alkyl,(C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, aryl, orheteroaryl; wherein any (C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl,(C₂-C₂₀)alkenyl, or (C₂-C₂₀)alkynyl of R₃ is optionally substituted withone or more (e.g 1, 2, 3, or 4) halo, hydroxy, mercapto oxo, thioxo,carboxy, (C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkoxycarbonyl, aryl, heteroaryl, orNR_(d)R_(e); wherein R_(d) and R_(e) are each independently hydrogen,(C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,(C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkoxycarbonyl aryl, or heteroaryl; andwherein any aryl or heteroaryl is optionally substituted with one ormore (1, 2, 3, or 4) halo, mercapto, hydroxy, oxo, carboxy, cyano,nitro, trifluoromethyl, trifluoromethoxy, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkanoyloxy, sulfo or (C₁-C₂₀)alkoxycarbonyl; or a salt thereof.

Other suitable compounds include for example compounds of the formulaR₄NCS wherein: R₄ is (C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, aryl, or heteroaryl; wherein any (C₁-C₂₀)alkyl,(C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, or (C₂-C₂₀)alkynyl of R₃ isoptionally substituted with one or more (e.g 1, 2, 3, or 4) halo,hydroxy, mercapto oxo, thioxo, carboxy, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl, aryl, heteroaryl, or NR_(f)R_(g); wherein R_(f)and R_(g) are each independently hydrogen, (C₁-C₂₀)alkyl,(C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl aryl, or heteroaryl; and wherein any aryl orheteroaryl is optionally substituted with one or more (1, 2, 3, or 4)halo, mercapto, hydroxy, oxo, carboxy, cyano, nitro, trifluoromethyl,trifluoromethoxy, (C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkanoyloxy, sulfo or(C₁-C₂₀)alkoxycarbonyl; or a salt thereof.

Other suitable compounds that comprise a carbon-selenium single bond ora carbon sulfur single bond include compounds of formula R₅—X—R₆wherein:

X is —S— or —Se—;

R₅ is (C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, aryl, or heteroaryl; and R₆ is hydrogen, (C₁-C₂₀)alkyl,(C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, aryl, orheteroaryl;

or R₅ and R₆ together with X form a heteroaryl;

wherein any (C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, or(C₂-C₂₀)alkynyl of R₅ or R₆ is optionally substituted with one or more(e.g 1, 2, 3, or 4) halo, hydroxy, mercapto oxo, thioxo, carboxy,(C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkoxycarbonyl, aryl, heteroaryl, orNR_(k)R_(m);

wherein R_(k) and R_(m) are each independently hydrogen, (C₁-C₂₀)alkyl,(C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl aryl, or heteroaryl; and

wherein any aryl or heteroaryl is optionally substituted with one ormore (1, 2, 3, or 4) halo, mercapto, hydroxy, oxo, carboxy, cyano,nitro, trifluoromethyl, trifluoromethoxy, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkanoyloxy, sulfo or (C₁-C₂₀)alkoxycarbonyl; or a salt thereof.

Specific and preferred values listed below for radicals, substituents,and ranges, are for illustration only; they do not exclude other definedvalues or other values within defined ranges for the radicals andsubstituents

Specifically, (C₁-C₂₀)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, 3-pentyl, or hexyl;(C₃-C₈)cycloalkyl can be cyclopropyl, cyclobutyl, cyclopentyl, orcyclohexyl; (C₁-C₂₀)alkoxy can be methoxy, ethoxy, propoxy, isopropoxy,butoxy, iso-butoxy, sec-butoxy, pentoxy, 3-pentoxy, or hexyloxy;(C₂-C₂₀)alkenyl can be vinyl, allyl, 1-propenyl, 2-propenyl, 1-butenyl,2-butenyl, 3-butenyl, 1-pentenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl,1-hexenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, or 5-hexenyl;(C₂-C₂₀)alkynyl can be ethynyl, 1-propynyl, 2-propynyl, 1-butynyl,2-butynyl, 3-butynyl, 1-pentynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl,1-hexynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, or 5-hexynyl;(C₁-C₂₀)alkanoyl can be acetyl, propanoyl or butanoyl;(C₁-C₂₀)alkoxycarbonyl can be methoxycarbonyl, ethoxycarbonyl,propoxycarbonyl, isopropoxycarbonyl, butoxycarbonyl, pentoxycarbonyl, orhexyloxycarbonyl; (C₂-C₂₀)alkanoyloxy can be acetoxy, propanoyloxy,butanoyloxy, isobutanoyloxy, pentanoyloxy, or hexanoyloxy; aryl can bephenyl, indenyl, or naphthyl; and heteroaryl can be furyl, imidazolyl,triazolyl, triazinyl, oxazoyl, isoxazoyl, thiazolyl, isothiazoyl,pyrazolyl, pyrrolyl, pyrazinyl, tetrazolyl, pyridyl, (or its N-oxide),thienyl, pyrimidinyl (or its N-oxide), indolyl, isoquinolyl (or itsN-oxide) or quinolyl (or its N-oxide).

Specifically, R₁ and R₂ can each independently be hydrogen,(C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, (C₂-C₂₀)alkynyl,aryl, heteroaryl, or NR_(a)R_(b); wherein R_(a) and R_(b) are eachindependently hydrogen, (C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl,(C₂-C₂₀)alkenyl, (C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkoxycarbonyl,(C₂-C₂₀)alkynyl, aryl, or heteroaryl; wherein any (C₁-C₂₀)alkyl,(C₃-C₈)cycloalkyl, (C₁-C₂₀)alkoxy, (C₂-C₂₀)alkenyl (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl, or (C₂-C₂₀)alkynyl of R₁, R₂, R_(a), and R_(b)is optionally substituted with 1 or 2 halo, hydroxy, mercapto, oxo,thioxo, carboxy, (C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkoxycarbonyl, aryl, orheteroaryl; and wherein any aryl or heteroaryl is optionally substitutedwith one or more halo, hydroxy, mercapto, carboxy, cyano, nitro,trifluoromethyl, trifluoromethoxy, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkanoyloxy, sulfo or (C₁-C₂₀)alkoxycarbonyl.

Specifically, R₁ and R₂ can each independently be hydrogen,(C₁-C₁₀)alkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl, aryl, or NR_(a)R_(b).

Specifically, R₁ and R₂ together with the carbon to which they areattached can form a 5 or 6 membered saturated or unsaturated ringcomprising carbon and optionally comprising 1 or 2 heteroatoms selectedfrom oxy (—O—), thio (—S—), or nitrogen (—NR_(c))—, wherein said ring isoptionally substituted with 1, 2, or 3 halo, hydroxy, oxo, thioxo,carboxy, (C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₁-C₂₀)alkoxy,(C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkoxycarbonyl, (C₂-C₂₀)alkenyl,(C₂-C₂₀)alkynyl, aryl, or heteroaryl; wherein R_(c) is hydrogen,(C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl, (C₂-C₂₀)alkynyl, aryl, heteroaryl; wherein any(C₁-C₂₀)alkyl, (C₃-C₂₀)cycloalkyl, (C₁-C₂₀)alkoxy, (C₂-C₂₀)alkenyl(C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkoxycarbonyl, or (C₂-C₂₀)alkynyl of R₁, R₂,and R_(c) is optionally substituted with one or more halo, hydroxy,mercapto, oxo, thioxo, carboxy, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl, aryl, or heteroaryl; and wherein any aryl orheteroaryl is optionally substituted with one or more halo, hydroxy,mercapto, carboxy, cyano, nitro, trifluoromethyl, trifluoromethoxy,(C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkanoyloxy, sulfo or (C₁-C₂₀)alkoxycarbonyl.

Specifically, R₁ and R₂ can each independently be NR_(a)R_(b); whereinR_(a) and R_(b) are each independently hydrogen, (C₁-C₂₀)alkyl,(C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl, (C₂-C₂₀)alkynyl, aryl, heteroaryl; wherein any(C₁-C₂₀)alkyl, (C₃-C₈)cycloalkyl, (C₂-C₂₀)alkenyl (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl, or (C₂-C₂₀)alkynyl is optionally substitutedwith one or more halo, hydroxy, mercapto, oxo, thioxo, carboxy, aryl, orheteroaryl; and wherein any aryl or heteroaryl is optionally substitutedwith one or more halo, hydroxy, mercapto, carboxy, cyano, nitro,trifluoromethyl, trifluoromethoxy, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkanoyloxy, sulfo or (C₁-C₂₀)alkoxycarbonyl.

Specifically, R₁ and R₂ can each independently be amino, (C₁-C₂₀)alkyl,(C₁-C₂₀)alkylamino, allylamino, 2-hydroxyethylamino, phenylamino, or4-thiazoylamino.

Specifically, R₁ and R₂ can each independently be amino, methyl,allylamino, 2-hydroxyethylamino, phenylamino, or 4-thiazoylamino.

A specific value for R₃ is (C₁-C₂₀)alkyl optionally substituted with oneor more halo, mercapto oxo, thioxo, carboxy, (C₁-C₂₀)alkanoyl,(C₁-C₂₀)alkoxycarbonyl, aryl, heteroaryl, or NR_(d)R_(e).

A specific value for R₃ is 2-aminoethyl, 2-amino-2-carboxyethyl, or2-acylamino-2-carboxyethyl.

A specific value for R₄ is aryl, optionally substituted with one or morehalo, mercapto, hydroxy, oxo, carboxy, cyano, nitro, trifluoromethyl,trifluoromethoxy, (C₁-C₂₀)alkanoyl, (C₁-C₂₀)alkanoyloxy, sulfo or(C₁-C₂₀)alkoxycarbonyl.

Specifically, R₅ is (C₁-C₁₀)alkyl, (C₃-C₆)cycloalkyl, (C₂-C₁₀)alkenyl,(C₂-C₁₀)alkynyl, aryl, or heteroaryl; and R₆ is hydrogen, (C₁-C₁₀)alkyl,(C₃-C₆)cycloalkyl, (C₂-C₁₀)alkenyl, (C₂-C₁₀)alkynyl, aryl, orheteroaryl.

Specifically, R₅ and R₆ together with X form a heteroaryl.

In one embodiment, the organic compound is not a polypeptide or proteinwith one or more mercapto (C—SH) groups.

In one embodiment, the organic compound is not a compound that includesone or more mercapto (C—SH) groups.

Preferred compounds are coenzyme A and DTT.

The compounds described hereinabove are available from commercialsources or can be prepared from commercially available startingmaterials using procedures that are known in the field of syntheticchemistry. For example, see Jerry March, Advanced Organic Chemistry, 4thed. Wiley-Interscience, John Wiley and Sons, New York, 1992.

In cases where compounds are sufficiently basic or acidic to form stablesalts, use of the compounds as salts in the methods of the invention maybe appropriate. Examples of suitable salts include organic acid additionsalts, for example, tosylate, methanesulfonate, acetate, citrate,malonate, tartarate, succinate, benzoate, ascorbate, α-ketoglutarate,and α-glycerophosphate salts. Suitable inorganic salts may also beformed, including hydrochloride, sulfate, nitrate, bicarbonate, andcarbonate salts.

Salts can be obtained using standard procedures well known in the art,for example by reacting a sufficiently basic compound with a suitableacid. Alkali metal (for example, sodium, potassium or lithium) oralkaline earth metal (for example calcium) salts can also be used.

When used in accord with the methods of the invention, the compoundsdescribed herein can be present in a luminescence reaction at anyeffective concentration. The optimum concentration of a given compoundwill depend on the luminescent reagent(s) employed, and on the specificconditions under which a given assay is carried out. However, suitableconcentrations can be determined using standard techniques that areavailable in the art.

Specifically, the compound can be present in a luminescence reaction ata concentration of at least about 0.01 μM, or at a concentration of atleast about 0.1 μM, e.g., at least about 0.1 mM. More specifically, thecompound can be present in the luminescence reaction at a concentrationin the range from about 0.1 μM to about 500 mM (inclusive), or in therange from about 0.001 μM, 0.01 μM, or 0.1 μM to about 250 mM(inclusive). Preferably, the compound is present at a concentration inthe range from about 0.001 μM, 0.01 μM, 0.1 μM, 1 μM or 10 μM to about100 mM (inclusive).

Specifically, the assay can be performed in the presence of purifiedenzymes, cell lystates or whole cells.

Specifically, the assay can be carried out in a solvent comprising atleast about 10% water. More specifically, the invention can be carriedout in a solvent comprising at least about 25% water, or at least about40% water.

VII. Kits

The present invention also provides kits for detecting the presence oractivity of one or more molecules which are reagents for a reaction,enhance or inhibit a reaction, or for detecting a condition, in a samplesuch as a sample including intact cells, a cell lysate, e.g., a lysatewhich is at least partially purified, or a cellular supernatant. In oneembodiment, the kit includes a derivative of the invention. For kits ofthe invention that include two or more of the following, a derivative ofluciferin or aminoluciferin, a derivative of a fluorophore, anothersubstrate, enzyme, or reaction mixture, each can be contained in aseparate container, some components may be combined in some containers,or they can be contained in a single container. The kit can optionallycomprise a buffer solution suitable for use in an assay, and thederivative or enzyme, and the buffer solution can optionally becontained in a single container. Additionally, the derivative and thebuffer solution can optionally be contained in a single container. Thekits can also optionally include a quenching agent for anonbioluminescent reaction.

IX. Exemplary Synthesis

A. Experimental Procedures for Luciferin Modifications

Included below are experimental procedures for syntheses of variousluciferin derivatives:

I. General procedures for synthesizing esters of luciferins

II. D-cysteine or D-cystine esters

III. Esters of luciferin methyl ether or luciferin-H

IV. Quinolinyl luciferin derivatives

V. Miscellaneous

I. General Procedures for Synthesizing Luciferin Esters

There are two ways of synthesizing esters of D-luciferin andderivatives, including luciferin-H and quinolinyl luciferin: a) bydirect cyclization of the corresponding cyano group with D-cysteine orD-cystine esters, and b) by CsF-promoted esterification with halogenatedorganic compounds, such as 3-iodomethyl pyridine hydriodide (picolinyliodide) or 3-iodopropanol.

A. Direct Cyclization

Under this category, two D-cysteine esters are used, one is D-cysteinemethyl ester and the other is D-cystine 2-hydroxyethyl ester. The latterrequires a reducing agent before cyclization.

i) D-Cysteine Methyl Ester

A derivative of 2-cyanobenzothiazole or 2-cyanoquinoline (1 equivalent)was dissolved in methanol and was degassed with a gentle stream ofnitrogen for 10 minutes. Meanwhile, D-cysteine methyl ester (1.5equivalents) was dissolved in water and its pH was adjusted to about 7.This solution was also degassed with a gentle stream of nitrogen for 5minutes. Then, the aqueous solution was added to above organic solutiondropwise and the resultant solution was stirred under nitrogen for 30minutes to a few hours depending on substrates. The reaction wasmonitored by TLC with methanol in dichloromethane. After the completionof the reaction, the mixture was filtered and was purified by HPLC.Typical HPLC conditions are the following.

Column: Dynamax 60 Å, 1 inch, 250 mm, reversed phase C-18 column

Mobile phase A: water or 100 mM ammonium acetate, pH 7, or 0.1% TFA inwater

Mobile phase B: acetonitrile

Flow rate: 20 mL/min

Detection wavelength: 300 nm

Gradient: from 100% A to 100% B, 30 minutes, and keep 100% B for 30minutes.

The purified compound was then characterized by NMR, MS, UV-Vis, andHPLC.

ii) D-Cystine 2-Hydroxyethyl Ester

The procedure of cyclization with D-cystine 2-hydroxyethyl ester is verysimilar to D-cysteine methyl ester except 1 equivalent oftris(2-carboxyethyl)phosphine hydroxhloride (TCEP) was added before pHadjustment.

B. CsF-Promoted Esterification

To a solution of luciferin or a derivative (1 equivalent) in DMF wasadded cesium fluoride (1.5 equivalents), followed by3-iodomethylpyridine hydriodide or 3-iodopropanol (1.5 equivalents).After the resultant mixture was stirred at room temperature for a fewhours, monitored by TLC with methanol in dichloromethane, the solutionwas concentrated down and water was added. The mixture was then filteredthrough a 0.2 μm filter and was subjected to HPLC purification usingsimilar condition described above.II. D-Cysteine or D-cystine esters1. Synthesis of 2-hydroxyethyl Ester of D-Cystine

To anhydrous ethylene glycol solution (10 mL) was charged D-cysteinehydrochloride monohydrate (1 g). A gentle stream of hydrochloride gaswas introduced to bubble the solution for 1 hour. The solution was thenlet stand over night. Cold isopropyl alcohol (12.5 mL) was added slowlyand the resultant mixture was put at −20° C. for 1 hour. The whiteprecipitates were then filtered, washed with cold isopropyl alcohol, andpumped by maintaining the suction of the filtrate cake. White crystals(595 mg) were obtained (yield: 52%).

¹H NMR (DMSO-d6): 8.6-88 ppm (broad, 6H), 4.35 ppm (broad singlet, 2H),4.20 ppm (m, 4H), 3.63 ppm (m, 4H), 3.05 ppm (broad singlet, 4H) ES⁺330.53 (M.W. 330.44)

2. Synthesis of Methyl Ester of D-Cysteine

To a methanol solution (100 mL) was charged D-cysteine hydrochloridemonohydrate (6 g). A gentle stream of hydrochloride gas was introducedto bubble the solution for 105 minutes. The solution was then stirredover night. Then the solvent was then removed under vacuum. The whiteresidue was dissolved in hot methanol (25 mL) and 25 mL of isopropylalcohol was added. The mixture was put on a rotavap to remove some ofthe methanol until white precipitates crashed out of the solution. Theprecipitates were put on ice briefly and then filtered under vacuum,washed with cold isopropyl alcohol, and pumped by maintaining thesuction of the filtrate cake.

¹H NMR (CD3OD): 4.35 ppm (t, 1H), 3.85 ppm (s, 3H), 3.10 ppm (d, 2H)

III. Luciferin Methyl Ether Esters

3. Synthesis of 2-hydroxyethyl Ester of Luciferin Methyl Ether

a. By 2-hydroxyethyl Ester of D-Cystine

2-cyano-6-methoxybenzothiazole (100 mg, 0.526 mmol) was dissolved inmethanol (20 mL) and the resultant solution was degassed with a gentlestream of nitrogen for 10 minutes.

To a solution of 2-hydroxyethyl ester of D-cysteine (87 mg, 0.263 mmol)in water (10 mL) was added tris(2-carboxyethyl)phosphine hydroxhloride(TCEP) (77 mg, 0.263 mmol). The pH of this solution was adjusted toabout 7 with 1 M potassium carbonate. The resultant solution was addedto aforementioned solution dropwise and the mixture was stirred for 30minutes. The precipitates were filtered out through a 0.2 μm filter andthe filtrate was purified by semi-preparative HPLC (see generalprocedure: Mobile phase A: water). 102 mg of pure compound was obtained(Yield: 52%).

¹H NMR (CD3CN): 8.0 ppm (d, 1H), 7.55 ppm (d, 1H), 7.20 ppm (dd, 1H),5.42 ppm (t, 1H), 4.25 ppm (m, 2H), 3.75 ppm (m, 4H)

MS: ES⁺ 338.58 (M.W. 338.40)

Extinction coefficient: 16,630 (at 325 nm in acetonitrile)

b. By Ethylene Glycol

To a solution of luciferin methyl ether (100 mg, 0.34 mmol) in anhydrousethylene glycol (4 mL) in a 5 mL Schlenk flask was introduced a gentlestream of hydrochloride gas. After 5 minutes of bubbling, the flask wassealed and put into an oil bath of 62° C. for 3 hours. The HCl bubblingwas repeated for another 5 minutes and put back into the oil bath foranother 1 hour. After the solution was cooled to room temperature, water(6 mL) was added and the resultant mixture was filtered and purifiedwith semi-preparative HPLC (see general procedure: Mobile phase A:water).

4. Synthesis of 2-hydroxy Ethyl Ester of Luciferin-H

This compound was synthesized in a similar way to procedure described insection 3a.

¹H NMR (CD3CN): 8.15 ppm (m, 2H), 7.60 ppm (m, 2H), 5.45 ppm (t, 1H),4.25 ppm (m, 2H), 3.75 ppm (m, 4H), 1.95 ppm (t, 1H)

MS: ES⁺ 308.58 (M.W. 308.38)

Extinction coefficient: 14,050 (at 294 nm in acetonitrile)

5. Synthesis of M-Picolinyl Ester of Luciferin Methyl Ether

To a solution of luciferin methyl ether (215 mg, 0.73 mmol) in DMF (35mL) was added cesium fluoride (170 mg, 11.1 mmol), followed by3-iodomethylpyridine hydriodide (380 mg, 1.1 mmol). After the resultantmixture was stirred at room temperature for 3 hours and twenty minutes,the solution was concentrated down to about 10 mL and 6 mL of water wasadded. The mixture was filtered through a 0.2 μm filter and wassubjected to HPLC purification (see general procedure: Mobile phase A:0.1% TFA in water).57 mg of pure compound was obtained.

¹H NMR (CD₃CN): 8.62 ppm (s, 1H), 8.54 ppm (d, 1H), 7.96 ppm (d, 1H),7.84 ppm (d, 1H), 7.56 ppm (d, 1H), 7.41 ppm (m, 1H), 7.20 ppm (dd, 1H)

MS: ES⁺ 385.77 (M.W. 385.46)

6. Synthesis of M-Picolinyl Ester of Luciferin

To a solution of luciferin mono potassium salt (51 mg, 0.16 mmol) in DMF(10 mL) was added cesium fluoride (24 mg, 0.16 mmol), followed by3-bromomethyl pyridine hydrobromide (44 mg, 0.17 mmol). After 16 hoursof reaction at room temperature, the solvent was stripped away undervacuum. The residue was then dissolved in 10 mL of 60% DMF in water. Theresultant solution was filtered through an 0.2 μm filter and wassubjected to HPLC purification (see general procedure: Mobile phase A:water). 8.6 mg of pure compound was obtained.

¹H NMR (CD3OD-D2O): 8.58 ppm (s, 1H), 8.50 ppm (d, 1H), 7.90 ppm (d, 1H,7.85 ppm (dd, 1H), 7.42 ppm (dd, 1H), 7.40 ppm (s, 1H), 7.10 ppm (dd,1H), 5.45 ppm (t, 1H), 5.25 ppm (s, 2H), 3.75 ppm (m, 2H)

MS: ES⁺: 371.65 (M.W. 371)

UV-Vis: 263 nm and 334 nm

7. Synthesis of M-Picolinyl Ester of Luciferin-H

This compound was synthesized and purified in a similar fashion to thesynthesis of m-picolinyl ester of luciferin methyl ether usingluciferin-H as starting material. 13 mg of pure compound was obtained.

¹H NMR (CD3CN-D2O): 8.60 ppm (s, 1H), 8.52 ppm (d, 1H), 8.10 ppm (m,2H), 7.85 ppm (m, 1H), 7.59 ppm (m, 2H), 7.42 ppm (m, 1H), 5.50 ppm (t,1H), 5.28 ppm (s, 2H), 3.79 ppm (m, 2H)

MS: ES⁺ 355.54 and 711.36 (dimer) (M.W. 355.43)

8. Synthesis of 3-Hydroxypropyl Ester of Luciferin Methyl Ether

This compound was synthesized in a similar way to m-picolinyl ester ofluciferin methyl ether using luciferin methyl ether and 3-iodopropanolas starting materials.

¹H NMR (CD3OD): 7.95 ppm (d, 1H), 7.55 ppm (d, 1H), 7.18 ppm (dd, 1H),5.41 ppm (t, 1H), 4.35 ppm (t, 2H), 3.92 ppm (s, 3H), 3.75 ppm (dd, 2H),3.65 ppm (t, 2H), 1.92 ppm (m, 2H)

MS: ES⁺: 352.70 (M.W. 352.43)

UV-Vis: 330 nm and 264 nm.

IV. Quinolinyl Luciferin Derivatives

9. Synthesis of 2-Hydroxy Ester of Quinolinyl Luciferin-H

A solution of 2-cyanoquinoline (50 mg, 0.32 mmol) in 1,4-dioxane (5 mL)was degassed for 5 minutes by passing through a gentle stream ofnitrogen gas.Cystine 2-hydroxyethyl ester hydrochloride (53 mg, 0.16 mmol) in water(5 mL) was treated with tris(2-carboxyethyl)phosphine hydrochloride (46mg, 0.16 mmol). The pH of this solution was adjusted to about 7 with 1 Mpotassium carbonate. It was then added dropwise to the solution of2-cyanoquinoline. The resultant solution was stirred at room temperaturefor 7.5 hours and was purified in a similar way to m-picolinyl ester ofluciferin (see general procedure: Mobile phase A: water). 13 mg of purecompound was obtained (yield: 26.7%)

¹H NMR (CD3-OD-D2O): 8.40 ppm (d, 1H), 8.15 ppm (d, 1H), 8.10 ppm (d,1H), 7.95 ppm (d, 1H), 7.82 ppm (m, 1H), 7.68 ppm (m, 1H), 5.53 ppm (t,1H), 4.25 ppm (m, 2H), 3.75 ppm (m, 2H), 3.65 pppm (m, 2H)

MS: ES⁺: 302.35, 324.47 (M.W. 302.35)

UV-Vis: 247 nm and 295 nm.

Extinction coefficient: 7,500 (at 288 nm in acetonitrile)

10. Synthesis of Quinolinyl Luciferin-H

a. 2-cyanoquinoline

To a solution of quinoline N-oxide (2 g, 13.8 mmol) was added benzoylchloride (2.6 mL, 22.0 mmol), followed by water (20 mL). Then, asolution of potassium cyanide (3.6 g, 55.1 mmol) in water (80 mL) wasadded slowly over a period of 15 minutes with vigorous stirring. Dioxane(15 mL) was added to help the solubility. After the mixture was stirredat RT for 2.5 hours, it was extracted with equal volume ofdichloromethane three times. The organic layer was dried over anhydroussodium sulfate and the solvent was removed under vacuum. The residue wasthen purified by flash chromatography with 30% of ethyl acetate inhexane. 2.1 g of product was obtained (yield: 100%).

NMR (CDCl3): 8.32 ppm (d, 1H), 8.18 ppm (d, 1H), 7.85 ppm (m, 2H), 7.65ppm (m, 2H)

ES⁺: 152.44 (M.W. 154.16)

b. Quinolinyl Luciferin-H

2-cyanoquinoline (51 mg, 0.33 mmol) was dissolved in 10 mL of methanoland the resultant solution was degassed with a gentle stream of nitrogenfor 10 minutes. Meanwhile D-cysteine hydrochloride monohydrate (90 mg,0.51 mmol) was dissolved in 5 mL of water and the pH of the resultantsolution was adjusted to about 7. It was then degassed with a gentlestream of nitrogen for 5 minutes. The aqueous solution was then added tothe organic solution and the resultant mixture was stirred undernitrogen for 4 hours. It was then purified by semipreparative HPLC (seegeneral procedure: Mobile phase A: 0.1% TFA in water).

NMR: characteristic peaks: 5.45 ppm (t, 1H), 3.68 ppm (m, 2H)

ES⁺: 257.83 (M.W. 256.28)

UV-Vis: 295 nm

Extinction coefficient: 28,530 (at 242 nm in methanol)

11. Synthesis of Naphthyl Luciferin Methyl Ether

Starting with commercially available 6-methoxy-2-naphthonitrile, thiscompound was synthesized in one step similar to quinolinyl luciferin-H.

NMR (CD3CN): 8.29 ppm (s, 1H), 8.05 ppm (dd, 1H), 7.94 ppm (d, 1H), 7.86ppm (d, 1H), 7.36 ppm (d, 1H), 7.25 ppm (dd, 1H), 5.35 ppm (t, 1H), 3.95ppm (s, 3H), 3.75 ppm (m, 2H)

ES⁺: 288.23 (M.W. 287.33)

UV-Vis: 253 nm, 279 nm, 315 nm, 339 nm.

12. Synthesis of Methyl Ester of 6′-Hydroxyquinolinyl Luciferin MethylEther

a. 6′-hydroxyquinolinyl Luciferin Methyl Ether

This compound was synthesized in a similar fashion to quinolinylluciferin-H starting with 2-cyano-6-methoxyquinoline and D-cysteine.

NMR (CD3CN): 8.35 ppm (d, 1H), 8.16 ppm (d, 1H), 8.03 ppm (d, 1H), 7.52ppm (dd, 1H), 7.38 ppm (d, 1H), 5.45 ppm (t, 1H), 3.97 ppm (s, 3H), 3.75ppm (m, 2H)

ES⁺: 289.33 (288.32)

UV-Vis: 343 nm, 329 nm, 261 nm.

Extinction coefficient

b. Methyl Ester of 6′-Hydroxyquinolinyl Luciferin Methyl Ether (881-92)

To a mixture of 3 mL of 40% KOH solution and 10 mL of ethyl ether on icewas added N-nitroso-N-methylurea (1 g, 9.7 mmol) in portions over aperiod of 5 minutes. Once the bubbling died down, the yellow top layerwas carefully decanted into an Erlenmyer flask with a few potassiumhydroxide pellets. The mixture was let stand on ice for 1 hour.

To a solution of 6-hydroxyquinolinyl luciferin methyl ether (19.5 mg,0.068 mmol) in 10 mL of anhydrous THF was added dropwise thediazomethane solution made above using a fire-polished glass pipet untilthe yellow color persisted. 5 drops of acetic acid was added to quenchthe excess diazomethane. The solvent was removed under reduced pressureand the residue was dissolved in 40% water in acetonitrile and waspurified by semipreparative HPLC (see general procedure: Mobile phase A:water).

NMR (CD3CN): 8.25 ppm (d, 1H), 8.14 ppm (d, 1H), 7.96 ppm (d, 1H), 7.45ppm (dd, 1H), 7.36 ppm (d, 1H), 5.42 ppm (t, 1H), 3.95 ppm (s, 3H), 3.80ppm (s, 3H), 3.65 ppm (m, 3H)

ES⁺: 303.49 (M.W. 302.35)

UV-Vis: 249 nm, 265=n, 326 nm, 341 nm

13. Synthesis of 6-hydroxyquinolinyl Luciferin (Standard QuinolinylLuciferin)

This compound was synthesized according to literature procedures.

14. Synthesis of 8-hydroxyquinolinyl Luciferin

This compound was synthesized in a similar way to quinolinyl luciferin-Hwith commercially available 2-cyano-8-hydroxyquinoline and D-cysteine asstarting materials.15. Synthesis of 6-aminoquinolinyl Luciferin

a. 6-amino-2-cyanoquinoline

This compound was synthesized according to a literature procedure (seeWO 03/096980 A2).

b. 6-aminoquinolinyl luciferin

6-amino-2-cyanoquinoline (100 mg, 0.59 mmol) was dissolved in 35 mL ofmethanol and the resultant solution was degassed with a gentle stream ofnitrogen for 10 minutes. Meanwhile, D-cysteine hydrochloride monohydrate(130 mg, 0.74 mmol) was dissolved in 15 mL of water and the resultantsolution was degassed with a gentle stream of nitrogen for 5 minutes.The aqueous solution was then added to the organic solution and theresultant mixture was stirred under nitrogen for 4 hours. The volume ofthe solution was reduced under vacuum and it was then purified bysemipreparative HPLC with and acetonitrile (see general procedure:Mobile phase A: 100 mM NH₄Ac, pH7).

NMR (CD3CN): 8.25 ppm (d, 1H), 7.95 ppm (m, 2H), 7.46 ppm (dd, 1H), 7.06ppm (s, 1H), 5.53 ppm (t, 1H), 3.96 ppm (m, 2H)

ES⁺: 274.67 (M.W. 273.31)

UV-Vis: 268 nm, 330 nm, and 373 nm.

Extinction coefficient: 15, 320 (at 224 nm in MeOH), 13,020 (at 274 nmin MeOH)

16. Synthesis of Quinolinyl Luciferin Benzyloxymethyl Ether

a. 2-cyano-6-benzyloxymethyl quinoline

To a solution of 2-cyano-6-hydroxyquinoline (100 mg) in 10 mL of acetonewas added potassium carbonate (122 mg). After stirring at RT for threeminutes, the solution was put on ice. Benzyl chloromethyl ether (122 μL)was added through a needle and the resultant solution was stirred on icefor 2 hours. The solvent was then removed under vacuum and the residuewas purified by flash chromatography with dichloromethane. 157 mg wasobtained.

NMR (CDCl₃): 8.18 ppm (d, 1H), 8.12 ppm (d, 1H), 7.65 ppm (d, 1H), 7.56ppm (dd, 1H), 7.45 ppm (d, 1H), 7.34 ppm (m, 5H), 5.44 ppm (s, 2H), 4.76ppm (s, 2H)

ES⁺: 293.89 (M.W. 290.11)

UV-Vis: 250 nm, 325 nm, and 336 nm.

b. Quinolinyl Luciferin Benzyloxymethyl Ether

To a solution of 2-cyano-6-benzyloxymethyl quinoline (75 mg, 0.259 mmol)in 10 mL of methanol was added D-cysteine hydrochloride monohydrate (55mg, 0.31 mmol). Triethylamine (44 μL) was added dropwise through aneedle. The solution became turbid in about one minute and 3 mL of waterwas added to make it clear. The resultant solution was stirred at RT for4 hours. Then, the solution was purified by HPLC (see general procedure:Mobile phase A: 0.1% TFA in water). 37.1 mg of product was obtained.

NMR (CD3CN): 8.28 ppm (d, 1H), 8.17 ppm (d, 1H), 8.03 ppm (d, 1H), 7.55ppm (m, 4H), 7.33 ppm (m 5H), 5.49 ppm (s, 3H), 5.44 ppm (t, 1H), 4.78ppm (s, 3H), 3.63 ppm (m, 2H)

ES⁺: 396.03 (M.W. 394.14)

UV-Vis: 256 nm, 324 nm and 336 nm

Extinction coefficient: 10,000 (at 323 nm in acetonitrile), 8,770 (at338 nm in acetonitrile)

V. Miscellaneous

17. Synthesis of Hydrazide of Luciferin Methyl Ether

a. N′-tert-butoxycarbonyl Hydrazide of Luciferin Methyl Ether

To a suspension solution of luciferin methyl ether (100 mg, 0.34 mmol)in anhydrous THF (5 mL) was added N,N′-diisopropylcarbodiimide (DIC)(130 μL, 0.84 mmol) and a solution of HOBt (110 mg, 0.72 mmol) inanhydrous THF (2 mL) through needles. A solution of tert-butyl carbazate(100 mg, 0.75 mmol) in anhydrous THF (3 mL) was then added. Theresultant mixture was stirred in an atmosphere of nitrogen for 29 hoursand the solvent was stripped away under vacuum. The residue was purifiedby flash chromatography with 1% methanol in dichloromethane. 139 mg ofproduct was obtained (100% yield).

¹H NMR (CD2Cl2): 8.30 ppm (broad, 1H), 8.00 ppm (d, 1H), 7.40 ppm (d,1H), 7.17 ppm (dd, 1H), 6.56 ppm (broad, 1H), 5.35 ppm (m, 1H), 3.90 ppm(s, 3H), 3.80 ppm (m, 2H), 1.48 ppm (s, 9H)

b. Hydrazide of Luciferin Methyl Ether

N′-tert-butoxycarbonyl hydrazide of luciferin methyl ether was treatedwith TFA (10 mL) in the presence of 6 drops of triisopropylsilane (TIS)for 70 minutes. TFA was then removed under vacuum and the residue wasdissolved in 10 mL of 1:1:1 mixture of water:acetonitrile:DMF. HPLCpurification condition was similar to the procedure for synthesis ofm-picolinyl ester of luciferin (see general procedure: Mobile phase A:0.1% TFA in water). 64 mg of pure compound was obtained.

¹H NMR (CD3OD): 7.98 ppm (d, 1H), 7.56 ppm (d, 1H), 7.19 ppm (dd, 1H),5.35 ppm (t, 1H), 3.90 ppm (s, 3H), 3.75 ppm (m, 2H)

ES⁺: 308.36 (M.W. 308.38)

UV-vis: 331 nm, 267 nm

Extinction coefficient: 6,000 (at 329 nm in acetonitrile-water mixture)

18. Synthesis of Hydrazide of Luciferin

This compound was synthesized in a similar way to the hydrazide ofluciferin methyl ether.

a. N′-tert-butoxycarbonyl Hydrazide of Luciferin

¹H NMR (CDCl3): 7.81 ppm (d, 1H), 7.18 ppm (d, 1H), 6.93 ppm (dd, 1H),5.20 ppm (m, 1H), 3.63 ppm (m, 2H), 1.33 ppm (s, 9H)

ES⁺: 396.75 (M.W. 394.35)

b. Hydrazide of Luciferin

¹H NMR (DMF-d7): 9.52 ppm (broad, 1H), 8.15 ppm (d, 1H), 7.73 ppm (d,1H), 7.33 ppm (dd, 1H), 5.47 ppm (t, 1H), 4.63 ppm (broad, 2H), 3.91 ppm(m, 2H)

MS: ES⁺: 295.53 (M.W. 294.36)

UV-vis: 148 nm and 293 nm

Extinction coefficient: 16,900 (at 327 nm in acetonitrile)

19. Synthesis of Luciferin 3-(4-phenylpiperazin-1-yl)methylbenzyl Ether

a. 2-cyano-6-(3-bromomethylbenzyloxy)benzothiazole

To a solution of 2-cyano-6-hydroxybenzothiazole (1.04 g) in 30 mL ofacetone was added potassium carbonate (1.63 g), followed byα,α′-dibromo-m-xylene (4.7 g). The resultant solution was stirred at RTfor 1 hour and the solvent was removed under reduced pressure. Theresidue was resuspended in dichloromethane and washed with water. Theaqueous phase was extracted with more dichloromethane (2×) and theorganic extracts were combined, dried over sodium sulfate, and purifiedby flash chromatography using dichloromethane. 1.945 g of product wasobtained.

¹H NMR (CDCl3): 8.15 ppm (d, 1H), 8.44 ppm (d, 1H), 8.40 ppm (m, 4H),7.20-7.30 ppm (m, 2H), 5.16 ppm (s, 2H), 5.52 ppm (s, 2H)

ES⁺: 362.60 (Br isotope effect seen) (M.W. 359.24)

b. 2-cyano-6-[3-(4-phenylpiperazin-1-yl)methylbenzyloxy]benzothiazole

To a solution of 2-cyano-6-(3-bromomethylbenzyloxy)benzothiazole (1 g,2.8 mmol) in 20 mL of dichloromethane was added 1-phenylpiperazine (1.7mL, 11.2 mmol) dropwise. The resultant solution was stirred at RT for 30minutes. Then, 20 mL of saturated sodium bicarbonate solution was added.After partition, the aqueous phase was extracted with 20 mL ofdichloromethane. The organic extracts were combined, washed with watertwice and dried over sodium sulfate. The solvent was removed and theresidue was purified by flash chromatography using 1% methanol indichloromethane. 594 mg of product was obtained.

¹H NMR (CDCl₃): 8.09 ppm (d, 1H), 7.47 ppm (broad, 1H), 7.43 ppm (d,1H), 7.37 ppm (m, 2H), 7.34 ppm (d, 1H), 7.30 ppm (m, 1H), 7.26 ppm (d,1H), 7.24 ppm (m, 1H), 6.92 ppm (d, 2H), 6.86 ppm (m, 1H), 5.18 ppm (s,2H), 3.20 ppm (m, 4H), 2.63 ppm (m, 4H)

c. Luciferin 3-(4-phenylpiperazin-1-yl)methylbenzyl ether

This compound was synthesized with 594 mg of2-cyano-6-[3-(4-phenylpiperazin-1-yl)methylbenzyloxy]benzothiazole and147 mg of D-cysteine hydrochloride monohydrate according to a proceduresimilar to 6-aminoquinolinyl luciferin except DMF was used as solventfor 2-cyano-6-[3-(4-phenylpiperazin-1-yl)methylbenzyloxy]benzothiazoledue to its poor solubility in methanol. The reaction mixture waspurified by HPLC (see general procedure: Mobile phase A: 0.1% TFA inwater). 282 mg of product was obtained.

¹H NMR (CD3CN): 7.95 ppm (d, 1H), 7.40-7.60 ppm (m, 5H), 7.25 ppm (m,3H), 7.26 ppm (t, 1H), 7.23 ppm (s, 2H), 4.27 ppm (s, 2H), 3.70 ppm (m,2H), 3.20-3.30 ppm (broad, 8H)

ES⁺: 546.03 (M.W. 544.69)

UV-vis: 268 nm and 328 nm

Extinction coefficient: 16,380 (at 324 nm in acetonitrile)

20. Synthesis of Luciferin o-trifluoromethylbenzyl Ether

a. 2-cyano-6-(2-trifluoromethylbenzyloxy)benzothiazole

To a solution of 2-cyano-6-hydroxybenzothiazole (100 mg, 0.568 mmol) in10 mL of acetone was added potassium carbonate (157 mg, 1.14 mmol),followed by 2-(trifluoromethyl)benzyl bromide (204 mg, 0.852 mmol). Themixture was then stirred at RT for about 5 hrs. The solvent was thenremoved under reduced pressure and the residue was dissolved indichloromethane. The resultant solution was then extracted withdichloromethane. The organic phase was dried over sodium sulfate andpurified by flash chromatography using dichloromethane. 148 mg ofproduct was obtained.

¹H NMR (CDCl₃): 8.12 ppm (d, 1H), 7.75 ppm (d, 2H), 7.60 ppm (t, 1H),7.48 ppm (t, 1H), 7.41 ppm (d, 1H), 7.34 ppm (dd, 1H), 5.28 ppm (s, 2H)

¹⁹F NMR (CDCl₃): 61 ppm

MS: ES⁺: 335.74 (M.W. 334.32)

b. Luciferin 2-trifluoromethylbenzyl ether

This compound was synthesized in a similar way to 6-aminoquinolinylluciferin with 150 mg of2-cyano-6-(2-trifluoromethylbenzyloxy)benzothiazole and 120 mg ofD-cysteine hydrochloride monohydrate. The mixture was purified HPLC (seegeneral procedure: Mobile phase A: 0.1% TFA in water). 55 mg of productwas obtained.

¹H NMR (CD3CN): 8.03 ppm (d, 1H), 7.82 ppm (d, 1H), 7.70 ppm (t, 1H),7.65 ppm (d, 1H), 7.58 ppm (t, 1H), 7.36 ppm (dd, 1H), 5.40 ppm (t, 1H),5.39 ppm (s, 2H), 3.75 ppm (d, 2H)

ES⁺: 440 (M.W. 438.44)

UV-vis: 267 nm and 325 nm

Extinction coefficient: 13,940 at 323 nm

21. Synthesis of Luciferin 2,4,6-Trimethylphenyl Ether

a. 2-cyano-6-(2,4,6-trimethylbenzyloxy)benzothiazole

This compound was synthesized in a similar way to2-cyano-6-(2-trifluoromethylbenzyloxy)benzothiazole withα²-chloroisodurene (100 mg, 0.852 mmol). 168 mg product was obtained.

¹H NMR (CDCl3): 8.18 ppm (d, 1H), 7.52 ppm (d, 1H), 7.30 ppm (dd, 1H),6.94 ppm (s, 2H), 5.09 ppm (s, 2H)

ES⁻: 264.10 (M.W. 263.78)

b. Luciferin 6-(2,4,6-trimethylbenzyl)ether

This compound was synthesized in a similar way to2-cyano-6-(2-trifluoromethylbenzyloxy)benzothiazole with2-cyano-6-(2,4,6-trimethylbenzyloxy)benzothiazole (80 mg, 0.303 mmol).It was then purified by HPLC (see general procedure: Mobile phase A:0.1% TFA in water). 82 mg of product was obtained.

¹H NMR (CD3CN): 7.98 ppm (d, 1H), 7.65 ppm (d, 1H), 7.22 ppm (dd, 1H),6.92 ppm (s, 2H), 5.40 ppm (t, 1H), 5.12 ppm (s, 2H), 3.75 ppm (d, 2H),2.32 ppm (s, 6H), 2.28 ppm (s, 3H)

ES⁺: 414 (M.W. 412.53)

UV-vis: 268 nm and 329 nm

Extinction coefficient: 18,240 at 323 nm

22. Synthesis of N-Methylephedrine-Linker-Luciferin (X═O, Y═H) orN-Methylephedrine-Linker-Luciferin (X═NH, Y═Cl)

These compounds were synthesized toward linking an enzyme-specificsubstrate to luciferin through a linker. In this case, the enzyme is aP450 isozyme, but in general, is useful with other enzymes as well.

Compound B was synthesized under Mitsunobu reaction conditions usingdiisopropylazodicarboxylate and triphosphine in THF. It was then reducedby NaBH₄ in methanol to produce compound C.Compound D (X═O for luciferin and X═NH for aminoluciferin) wassynthesized separately with corresponding benzothiazole andp-nitrophenyl chloroformate. It was then coupled to compound C underbasic condition and a standard D-cysteine cyclization generated theproduct as shown above.C. Syntheses for Representative Luciferin DerivativesSyntheses for luciferin derivatives described in some of the examplesabove are provided below.I. Bioluminogenic MAO SubstratesA.

6-(3-Dimethylaminopropoxy)luciferin

Synthesis of 6-(3-dimethylaminopropoxy)-2-cyanobenzothiozole

The mixture of 6-hydroxy-2-cyanobenzothiozole (0.311 g, 1.77 mmol),3-chloropropyldimethylamine hydrochloride (0.36 g, 2.27 mmol), potassiumcarbonate (0.63 g, 4.57 mmol) and sodium iodide (0.034 g) in acetone (30ml) was heated to reflux overnight. Upon cooling to room temperature,the insoluble solid was removed by filtration. The compound was purifiedby flash chromatography using methylene chloride/methanol (96:4) aseluent in a yield of 86% (0.387 g).

¹H NMR (CD₂Cl₂): 7.98 (d, J=9.3 Hz, 1H), 7.32 (d, J=2.4 Hz, 1H), 7.16(dd, J=9.0 Hz, J=2.1 Hz, 1H), 4.04 (t, J=6.4 Hz, 2H, OCH₂), 2.36 (t,J=7.2 Hz, 2H, NCH₂), 2.13 (s, 6H, CH₃), 1.90 (m, 2H, CH₂).

Synthesis of MAO-1

To the solution of dimethylamine (0.235 g, 0.90 mmol) and D-cystine(0.158 g, 0.90 mmol) in methanol (5 ml), CH₂Cl₂ (1 ml) and H₂O (1 ml)was added K₂CO₃ (0.125 g, 0.90 mol). The mixture was stirred at roomtemperature for 5 min and then neutralized to slightly acidic condition.After removal of organic solvent, the product was purified by HPLC using0.1% TFA water/acetonitrile as eluent.

¹H NMR (CD₂Cl₂): 7.90 (d, 1H), 7.23 (s, 1H), 7.04 (d, 1H), 5.37 (t, 1H,CHCOO), 4.10 (t, 2H, OCH₂), 3.79 (m, 2H, CH₂), 3.32 (t, 2H, CH₂N), 2.97(s, 6H, CH₃), 2.30 (m, 2H, CH₂). MS (ES): m/e (M+), 365

B.

6-Aminopropoxyluciferin, 6-(3-Dimethylaminopropoxy)luciferin

The compound MAO-3 was synthesized by employing the similar method usedfor the synthesis of MAO-1. Except that amino group was protected byt-BOC and de-protected after alkylation of6-hydroxyl-2-cyanobenzothiozole. The final compound was precipitated outfrom the reaction solution and collected by filtration, washed withwater and methanol.

¹H NMR (d6-DMSO/TFA): 8.04 (d, 1H), 7.75 (s, 1H), 7.19 (d, 1H), 5.40 (t,1H, CHCOO), 4.08 (t, 2H, OCH₂), 3.6-3.8 (m, 2H, CH₂), 3.0 (m, 2H, CH₂N),2.05 (m, 2H, CH₂). MS (ES): m/e (M+), 337. λ_(max) 328 nm, ε_(max)15,500 cm⁻¹M⁻¹ in water.

C.

5-fluoro-6-aminopropoxy-luciferin

The compound MAO-4 was synthesized by employing the similar method forthe synthesis of MAO-3.

¹H NMR (d6-DMSO/TFA): 8.07 (d, 1H), 7.97 (d, 1H), 5.41 (t, 1H, CHCOO),4.24 (t, 2H, OCH₂), 3.6-3.8 (m, 2H, CH₂), 3.0 (m, 2H, CH₂N), 2.08 (m,2H, CH₂). MS (ES): m/e (M+1), 355λ_(max) 326 nm, ε_(max) 15,600 cm⁻¹M⁻¹in water.

D.

6-methylaminopropoxy-luciferin

The compound MAO-5 was synthesized by employing the similar method usedfor the synthesis of MAO-3.

¹H NMR (d6-DMSO/TFA): 8.04 (d, 1H), 7.74 (d, 1H), 7.19 (dd, 1H), 5.40(t, 1H, CHCOO), 4.16 (t, 2H, OCH₂), 3.6-3.8 (m, 2H, CH₂), 3.07 (m, 2H,CH₂N), 2.59 (t, 3H, NCH₃), 2.07 (m, 2H, CH₂). MS (ES): m/e (M+1), 351.λ_(max) 328 nm, □_(max) 17,500 cm⁻¹M⁻¹ in water

E.

5-fluoro-6-methylaminopropoxy-luciferin

The compound MAO-6 was synthesized by employing the similar method usedfor the synthesis of MAO-3.

¹H NMR (d6-DMSO/TFA): 8.08 (d, 1H), 7.98 (d, 1H), 5.41 (t, 1H, CHCOO),4.24 (t, 2H, OCH₂), 3.6-3.8 (m, 2H, CH₂), 3.08 (m, 2H, CH₂N), 2.59 (t,3H, NCH₃), 2.11 (m, 2H, CH₂). MS (ES): m/e (M+1), 369

F.

6-(3-methylaminopropoxy)quinolinyl-luciferin

The compound MAO-7 was synthesized by using 6-hydroxyl-2-cyanoquinolineas starting material and by employing the similar method used for thesynthesis of MAO-3.

¹H NMR (d6-DMSO/TFA): 8.37 (d, J=8.7 Hz), 8.13 (d, J=9.2 Hz, 1H), 8.02(d, J=9.0 Hz, 1H), 7.44-7.54 (m-overlap, 2H), 5.45 (dd, J=8.10 Hz,J=8.10 Hz, 1H, CH—COOH), 4.25 (t, J=6.0 Hz, 2H, OCH₂), 3.5-3.8 (m, 2H,SCH₂), 3.17 (m, 2H, NCH₂), 2.63 (m, 3H, CH₃), 2.15 (m, 2H, CH₂). MS(ES): m/e (M+1), 346. λ_(max)(nm)/ε_(max) (cm⁻¹M⁻¹): 259/30,600;326/10,400; 342/10,100 in water.

G.

6-(3-dimethylaminopropoxy)quinolinyl-luciferin

The compound MAO-8 was synthesized by employing the similar method usedfor the synthesis of MAO-1.

¹H NMR (d6-DMSO/TFA): 8.28 (d, J=8.4 Hz), 8.05 (d, J=8.7 Hz, 1H), 7.93(d, J=10.0 Hz, 1H), 7.94-8.04 (m-overlap, 2H), 5.07 (t, J=9.30 Hz, 1H,CH—COOH), 4.15 (t, J=6.6 Hz, 2H, OCH₂), 3.35-3.62 (m, 2H, SCH₂), 2.40(m, 2H, NCH₂), 2.16 (s, 6H, CH₃), 1.92 (m, 2H, CH₂). MS (ES): (M+1)/e,359. λ_(max) (nm)/ε_(max) (cm⁻¹M⁻¹): 258/33,200; 326/10,700; 340/10,700in water.

H.

6-(3-aminopropoxy)quinolinyl-luciferin

The compound MAO-9 was synthesized by employing the similar method usedfor the synthesis of MAO-3.

¹H NMR (d₆-DMSO/TFA): 8.35 (d, J=8.7 Hz), 8.10 (d, J=8.4 Hz, 1H), 7.99(d, J=8.7 Hz, 1H), 7.4-7.5 (m-overlap, 2H), 5.07 (dd, J=8.40 Hz, J=8.4,1H, CH—COOH), 4.23 (t, J=6.0 Hz, 2H, OCH₂), 3.50-3.54 (m, 2H, SCH₂), 3.0(m, 2H, NCH₂), 2.08 (m, 2H, CH₂). MS (ES): (M+1) m/e, 332. λ_(max)(nm)/ε_(max) (cm⁻¹M⁻¹): 260/31,600; 327/11,200; 341/11,800 in water.

I.

6-(3-dimethylamino-3-butoxy)luciferin

The compound MAO-10 was synthesized by employing the similar method usedfor the synthesis of MAO-1.

¹H NMR (d6-DMSO): 8.07 (d, 1H), 7.78 (s, 1H), 7.20 (dd, J=9.0 Hz, J=2.1Hz, 1H), 5.41 (t, 1H, CHCOO), 4.0-4.3 (m, 2H, CH₂), 3.6-3.8 (m, 2H,SCH₂), 2.73 (m, 6H, NCH₃), 1.8-2.4 (m, 2H, CH₂), 1.31 (d, 3H, CH₃). MS(ES): (M+1) m/e, 380. λ_(max) 327 nm, □_(max) 14,700 cm⁻¹M⁻¹ in water.

J.

Methyl 6-(3-aminopropoxy) luciferin ester

Synthesis of methyl 6-(t-BOC-3-aminopropoxy) luciferin ester

To the solution of 2-cyano-6-(t-BOC-3-aminopropoxy)benzothiazole (0.27g, 0.82 mmol) and D-cysteine methyl ester (0.20 g, 1.05 mmol) in 10 mlof methanol was added TEA (0.11 g, 0.15 ml). The reaction mixture wasstirred for 5 minutes. 20 ml of methylene chloride and 20 ml of waterwere added. The mixture was extracted three times with methylenechloride and the combined organic layer was dried over magnesiumsulfate. The compound was purified by flash chromatography usingmethylene chloride/ethyl acetate (95:5 to 90:10) as eluent in a yield of60%.

¹H NMR (CD₂Cl₂): 8.0 (d, J=9.0 Hz, 1H), 7.39 (d, J=2.4 Hz, 1H), 7.16(dd, J=9.3 Hz, J=2.1 Hz, 1H), 5.35 (t, J=9.3 Hz, 1H, CHCOO), 4.12 (t,2H, OCH₂), 3.6-3.8 (m, 2H, SCH₂), 3.31 (q, 2H, CH₂N), 2.02 (m, 2H, CH₂),1.42 (s, 9H, CH₃). MS (ES): m/e (M+1), 452.

Synthesis of MAO-11

The solution of TFA (1 ml) and tri-isopropylsilane (3 ul) in 10 ml ofmethylene chloride was added to methyl 6-(t-BOC-3-aminopropoxy)luciferin ester (0.23 g, 0.488 mmol) at 0° C. The resultant mixture wasstirred at 0° C. for 1 hour. 30 ml of ether and 20 ml of methanol wereadded and the solvent was then removed by evaporation. The residue waspurified by flash chromatography using methylene chloride/methanol(95:5) as eluent in a yield of 56%.

¹H NMR (d6-DMSO): 8.06 (d, J=8.7 Hz, 1H), 7.75 (d, J=2.4 Hz, 1H), 7.20(dd, J=9.0 Hz, J=2.1 Hz, 1H), 5.51 (dd, 1H, CHCOO), 4.08 (t, 2H, OCH₂),3.6-3.9 (m, 5H, OCH₃+CH₂), 2.99 (m, 2H, CH₂N), 2.03 (m, 2H, CH₂). MS(ES): m/e (M+1), 352. λ_(max) 331 nm, ε_(max) 17,100 cm⁻¹M⁻¹ in water.

K.

Methyl 6-(3-methylaminopropoxy)quinolinyl-luciferin ester

Synthesis of 6-(3-t-BOC-3-methylaminopropoxy)quinolinyl-luciferin

The compound was made by employing the similar method used for thepreparation of MAO-3.

¹H NMR (d6-DMSO): 8.33 (d, 1H), 8.08 (d, 1H), 7.97 (d, 1H), 7.4-7.5 (m,2H), 5.51 (dd, 1H, CHCOO), 4.12 (t, 2H, OCH₂), 3.6-3.9 (m, SCH₂), 3.37(t, 2H, NCH₂), 1.99 (m, 2H, CH₂), 1.30 (s, br, 9H, CH₃). MS (ES): m/e(M+1), 446

Synthesis of methyl 6-(3-t-BOC-3-methylaminopropoxy)quinolinyl-luciferinester

To the solution of 6-(3-t-BOC-3-methylaminopropoxy)quinolinyl-luciferin(0.651 g, 1.418 mmol) in 15 ml of THF was added freshly-makingdiazomethane till the solution became yellow. The resultant mixture wasstirred for another 10 min, acetic acid was added and then 20 ml ofwater was added. The resultant mixture was extracted three times withmethylene chloride and dried over magnesium sulfate. The product waspurified by flash chromatography using methylene chloride/ethyl acetate(100/0 to 90/10) as eluent in a yield of 95%.

¹H NMR (d6-DMSO): 8.35 (d, 1H), 8.06 (d, 1H), 7.98 (d, 1H), 7.4-7.5 (m,2H), 5.51 (dd, 1H, CHCOO), 4.06 (t, 2H, OCH₂), 3.71 (s, OCH₃), 3.6-3.9(m, SCH₂), 3.38 (t, 2H, NCH₂), 2.0 (m, 2H, CH₂), 1.25 (s, br, 9H, CH₃).MS (ES): m/e (M+1), 461

Synthesis of MAO-12

The compound MAO-12 was prepared by deprotection of methyl6-(3-t-BOC-3-methylaminopropoxy)quinolinyl-luciferin ester by employingthe similar method used for the preparation of MAO-11.

¹H NMR (d6-DMSO): 8.36 (d, J=8.7 Hz), 8.10 (d, J=8.4 Hz, 1H), 7.99 (dd,J=8.1 Hz, Hz, J=2.4 Hz, 1H), 7.45 (dd, J=8.4 Hz, J=2.7 Hz, 1H), 7.4s (s,1H), 5.53 (dd, J=8.40 Hz, J=8.4 Hz, 1H, CH—COOH), 4.23 (t, J=6.0 Hz, 2H,OCH₂), 3.74 (s, 3H, CH₃), 3.50-3.73 (m, 2H, SCH₂), 3.09 (m, 2H, NCH₂),2.60 (s, br, 3H, NCH₃), 2.11 (m, 2H, CCH2). MS (ES): m/e (M+1), 361.λ_(max) (nm)/ε_(max) (cm⁻¹M⁻¹): 259/29,400; 326/9,800; 339/10,000 inwater.

II. Fluorogenic MAO Substrates

A.

7-(4-methyl-3-dimethylaminopropoxy)coumarin

Compound MAO-F1 was prepared by employing the similar method used forpreparation of MAO-1 precursor by alkylation of 7-(4-methyl-3-propoxy)coumarin with 3-chlorodimethylamino hydrochloride under basic condition.The compound was purified by flash chromatography using methylenechloride/methanol (90:10) as eluent in yield of 21%.

¹H NMR (d6-DMSO): 7.70 (d, 1H), 6.9-7.0 (m, 2H), 6.19 (s, 1H), 4.11 (t,2H, OCH₂), 2.3-2.5 (m, 5H, NCH₂+CH₃), 2.18 (s, 6H, NCH₃), 1.88 (m, 2H,CH₂). λ_(max) 381 nm, ε_(max) 16,100 cm⁻¹M⁻¹ in pH 7.5 buffer.

B.

7-(4-methyl-3-aminopropoxy)coumarin

Compound MAO-F2 was employed the similar method used for preparation ofMAO-F1 by alkylation of coumarin with t-BOC protectedaminopropylchloride, and the de-protection under acidic condition gavethe desired product.

¹H NMR (d6-DMSO): 7.68 (d, 1H), 6.9-7.0 (m, 2H), 6.21 (s, 1H), 4.28 (t,2H, OCH₂), 2.95 (m, 2H, NCH₂), 2.01 (m, 2H, CH₂). λ_(max) 384 nm,ε_(max) 15,500 cm⁻¹M⁻¹ in pH 7.5 buffer.

C.

7-(4-methyl-3-N-methylaminopropoxy)coumarin

Compound MAO-F3 was prepared by employing the similar method used forthe synthesis of MAO-F2.

D.

1-methoxy-6-aminopropoxyfluorescein lactone

Synthesis of Mono-t-BOC-Amidopropyl Fluorescein

To the solution of fluorescein (1.0 g, 3.0 mmol),t-BOC-amidopropylbromide (0.86 g, 3.61 mmol) and potassium carbonate(0.50 g, 3.62 mmol) in DMF/acetone (1:1) was heated to reflux overnight. TLC indicated that three products, mono-ether, mono-ester anddiester, were produced which was confirmed by MS. The products wereroughly purified by flash chromatography to remove fluorescein. Thecrude products (0.83 g) obtained were dissolved in 2N sodium hydroxideaqueous solution and heated to reflux for 3 hours. The solution wasacidify and extracted three times with ethyl acetate. The combinedorganic layer was dried over magnesium sulfate. After removal of thesolvent, the compound was purified by flash chromatography usingheptane/ethyl acetate (7/3 to 1/1) as eluent in a yield of 23%.

¹H NMR (CD₂Cl₂): 7.98 (d, 1H), 7.66 (m, 2H), 7.22 (d, 1H), 6.90 (s, br,2H), 6.67 (d, 2H), 6.55 (d, 2H), 4.0 (t, 2H, OCH₂), 3.05 (q, 2H, NCH₂),1.98 (m, 2H, CH₂), 1.38 (s, 9H, CH₃). MS (ES): m/e (M+1), 490.

Synthesis of Methoxy-t-BOC-Amidopropyl Fluorescein

To the solution of mono-t-BOC-amidopropyl fluorescein (0.4 g, 0.82 mmol)in THF/benzene (5 ml/15 ml) was added Ag₂O (0.56 g, 2.45 mmol) andmethyl iodide (0.46 g, 3.26 mmol). The resultant mixture was heated inthe dark overnight. Upon cooling to room temperature, the solid wasremoved by filtration. The solvent of filtrate was removed under reducedpressure and the product was purified by flash chromatography usingheptane/ethyl acetate (7/3) as eluent in a yield of 47%.

¹H NMR (CD₂Cl₂): 7.95 (d, 2H), 7.66 (m, 2H), 7.14 (d, 2H), 6.78 (s, br,2H), 6.55-6.72 (m, 2H), 4.03 (t, 2H, OCH₂), 3.81 (s, 3H, OCH₃), 3.25 (q,2H, NCH₂), 1.98 (m, 2H, CH₂), 1.40 (s, 9H, CH₃). MS (ES), m/e (M+1),504.

Synthesis of FMAO-4

The compound was made by de-protection of t-BOC as used for thesynthesis MAO-11.

¹H NMR (CD₂Cl₂):

MS (ES), m/e (M+1), 404

III. Bioluminogenic FMO Substrates

A.

6-(2-phenylthioethoxy)-luciferin

Synthesis of 2-chloroethyl phenyl sulfide

Benzenethiol (15.0 g, 0.136 mol) was added to the solution of sodiumethoxide (0.20 mol) in 200 ml of anhydrous ethanol, and the mixture wasstirred at room temperature for 15 minutes and then added to thesolution of 1-bromo-2-chloroethane in 100 ml of ethanol. The resultantmixture was stirred at room temperature for 3 hours and poured into 300ml of water. The mixture was extracted three times with ether and thecombined organic layer was dried over magnesium sulfate. After removalof the solvent, the compound was purified by flash chromatography usingheptane/ethyl acetate (100 to 97/3) as eluent in a yield of 74%.

¹H NMR (CD₂Cl₂): 7.2-7.5 (m, 5H), 3.64 (t, 2H), 3.23 (t, 2H)

Synthesis of 2-cyano-6-(2-phenylthioethoxy)benzothiozole

The compound was synthesized by employing the similar method for thepreparation of MAO-1 precursor and purified by flash chromatographyusing heptane/ethyl acetate (90:10) as eluent in a yield of 29%.

¹H NMR (CD₂Cl₂): 8.09 (d, 1H), 7.45 (d, 2H), 7.34 (t, 2H), 7.3 (s, 1H),7.28 (d, 1H), 7.22 (dd, 1H), 4.22 (t, 2H), 3.39 (t, 2H)

Synthesis of FMO-1

The compound was synthesized by employing the similar method for thepreparation of MAO-1 and purified by flash chromatography usingmethylene chloride/methanol (95:5) as eluent in a yield of 50%.

¹H NMR (d₆-DMSO): 8.0 (d, 1H), 7.71 (s, 1H), 7.40 (d, 2H), 7.32 (t, 2H),7.20 (t, 1H), 7.12 (d, 1H), 5.39 (t, 1H, CHCOO), 4.24 (t, 2H, OCH₂),3.6-3.8 (m, 2H, CH₂), 3.40 (t, 2H, SCH₂). MS (ES) m/e (M+1): 417.λ_(max) 328 nm, ε_(max) 19,900 cm⁻¹M⁻¹ in water

B.

6-(2-ethylthioethoxy)-luciferin

The compound FMO-2 was synthesized by employing the similar method forthe preparation of FMO-1.

¹H NMR (d₆-DMSO): 8.01 (d, 1H), 7.78 (s, 1H), 7.17 (d, 1H), 5.39 (t, 1H,CHCOO), 4.22 (t, 2H, OCH₂), 3.6-3.8 (m, 2H, CH₂), 2.91 (t, 2H, SCH₂),2.64 (q, 2H, CH₂), 1.23 9t, 2H, CH₃). MS (ES) m/e (M+1): 370. λ_(max)328 nm, ε_(max) 17,600 cm⁻¹M⁻¹ in methanol.

C.

6-(2-ethylsulfoxylethoxy)-luciferin

Synthesis of 2-cyano-6-(2-ethylsulfoxylethoxy)benzothiozole

To the solution of 2-cyano-6-(2-ethylthioethoxy)benzohiozole (0.34 g,1.29 mmol) in 10 ml of methylene chloride was added m-CPBA (0.33 g, 67%,1.30 mmol). The resultant mixture was stirred for 1 hour. The productwas purified by flash chromatography using ethlyl acetate/methanol(90/10) as eluent in a yield of 87%.

¹H NMR (CD₂Cl₂): 8.11 (d, 1H), 7.47 (d, 2H), 7.26 (d, 1H), 4.54 (m, 2H,OCH₂), 3.0-3.3 (m, 2H, CH₂SO), 2.88 (m, 2H, CH₂CH₃), 1.40 (t, 3H, CH₃).MS (ES), m/e (M+1), 282

Synthesis of FMO-3

The compound was synthesized by employing the similar method used forthe preparation of FMO-1.

¹H NMR (d₆-DMSO): 8.04 (d, 1H), 7.81 (s, 1H), 7.20 (d, 1H), 5.41 (t, 1H,CHCOO), 4.45 (m, 2H, OCH₂), 3.6-3.8 (m, 2H, CH₂), 3.0-3.4 (m, 2H,SOCH₂), 2.6-3.0 (m, 2H, CH₂CH₃), 1.23 (t, 2H, CH₃). MS (ES) m/e (M+1):385. λ_(max) 328 nm, ε_(max) 18,000 cm⁻¹M⁻¹ in methanol.

D.

6-(3-methylamino-1-butoxy)luciferin

Synthesis of 1-methyl-3-hydroxypropylmethyl-t-BOC-amide

To the solution of t-BOC anhydride (15.8 g, 0.0724 mol) in 150 ml ofanhydrous methylene chloride was added the solution of3-methylamino-1-butanol (6.22 g, 0.0604 mol) and (7.33 g, 0.0724 mol) in60 ml of methylene chloride at 0° C. The resultant mixture was stirredfor 3 hours. 200 ml of methylene chloride was then added. The mixturewas washed three times with water and the combined organic layer wasdried over magnesium sulfate. After removal of solvent, the product waspurified by flash chromatography using heptane/ethyl acetate as eluent(70/30 to 40/60) in a yield of 74%.

¹H NMR (CD₂Cl₂): 3.35 (m, 1H, CH), 2.8-3.5 (m, 2H, OCH₂), 2.63 (s, 3H,NCH₃), 1.65 (m, 2H, CH₂), 1.45 (s, 9H, CH₃), 1.18 (d, 3H, CH₃). MS (ES)m/e (M+1): 204

Synthesis of 1-methyl-3-bromopropylmethyl-t-BOC-amide

To the solution of 1-methyl-3-hydroxypropylmethyl-t-BOC-amide (9.01 g,0.0444 mol) and carbon tetrabromide (17.67 g, 0.0533 mol) in 120 ml ofanhydrous methylene chloride was added triphenylphosphine (14.0 g,0.0533 mol) at 0° C. The resultant mixture was stirred overnight. Afterremoval of solvent, the product was purified by flash chromatographyusing heptane/ethyl acetate as eluent (90/10) in a yield of 37%.

¹H NMR (CD₂Cl₂): 4.32 (m, 1H, CH), 3.34 (t, 2H, BrCH₂), 2.69 (s, 3H,NCH₃), 1.8-2.2 (m, 2H, CH₂), 1.45 (s, 9H, CH₃), 1.16 (d, 3H, CH₃)

Synthesis of 2-cyano-6-(1-methyl-3-propylmethyl-t-BOC-amide)benzothiozole

The similar procedure was employed by the method for the synthesis ofprecursor of MAO-1.

Synthesis of 6-(1-methyl-3-propylmethyl-t-BOC-amide) luciferin

The similar procedure was employed by the method for the synthesis ofMAO-1.

¹H NMR (CD₂Cl₂): 7.88 (d, 1H), 7.24 (s, 1H), 7.03 (d, 1H), 5.24 (t, 1H,CHCOO), 4.36 (s, br, 1H, CH), 3.91 (t, 2H, OCH₂), 3.67 (d, 2H, SCH₂),2.63 (s, 3H, NCH₃), 1.7-2.0 (m, 2H, CH₂), 1.25 (s, br, 9H, CH₃), 1.05(d, 3H, CH₃). MS (ES), m/e (M+2): 467

Synthesis of FMO-4

The compound FMO-4 was made by de-protection of t-BOC luciferin with TFAacid as described in the preparation of MAO-11.

¹H NMR (d6-DMSO): 8.04 (d, 1H), 7.76 (d, 1H), 7.19 (dd, 1H), 5.38 (t,1H, CHCOO), 4.18 (m, 1H, OCH2), 3.6-3.8 (m, 2H, SCH₂), 3.38 (m, 1H,NCH), 2.49 (s, 3H, NCH₃), 1.8-2.3 (m, 2H, CH₂), 1.24 (d, 3H, CH₃). MS(ES), m/e (M+2): 367. λ_(max) 326 nm, ε_(max) 16,800 cm⁻¹M⁻¹ in water.

E.

6-(3-methylamino-1-butoxy)quinolinyl-luciferin

The compound FMO-5 was synthesized by employing the similar method forthe preparation of FMO-4.

¹H NMR (d6-DMSO): 8.34 (d, 1H), 8.10 (d, 1H), 7.99 (d, 1H), 7.47 (m,overlap, 2H), 5.40 (t, 1H, CHCOO), 4.27 (m, 1H, OCH2), 3.5-3.6 (m, 2H,SCH₂), 3.38 (m, 1H, NCH), 2.60 (s, 3H, NCH₃), 1.9-2.3 (m, H, CH₂), 1.28(d, 3H, CH₃). MS (ES), m/e (M+2): 361. λ_(max) 327 nm, ε_(max) 9,970cm⁻¹M⁻¹ in water.

IV. Bioluminogenic GST/Glutathione substrates

A.

6-(2-nitro-4-trifluoromethyl-phenoxy)quinolinyl-luciferin

Synthesis of 2-cyano-6-(2-nitro-4-trifluoromethyl-phenoxy) quinoline

The mixture of 2-cyano-6-hydroxyquinoline (0.50 g, 2.94 mmol),2-nitro-4-trifluoromethylbenzene chloride (0.67 g, 2.94 mmol) andpotassium carbonate (0.41 g, 2.97 mmol) in 30 ml of DMSO was heated to100° C. for 30 min. Upon cooling to room temperature, the mixture waspoured into 30 ml of cold water and exacted three times with methylenechloride. The combined organic layer was washed with water and driedover magnesium sulfate. The product was purified by flash chromatographyusing heptane/methylene chloride (1:2) as eluent in a yield of 35%.

¹H NMR (CD₂Cl₂): 8.36 (d, 1H), 8.25 (dd, 1H), 7.94 (dd, 1H), 7.75 (d,1H), 7.67 (dd, 1H), 7.43 (d, 1H), 7.31 (d, 1H). MS (ES) m/e (M+2): 361.

Synthesis of GST-3

The compound was synthesized by employing the similar method for thepreparation of MAO-1 and was purified by flash chromatograph usingmethylene chloride/methanol (95:5) as eluent in a yield of 20%.

¹H NMR (d₆-DMSO): 8.55 (s, 1H), 8.42 (d, 1H), 8.19 (m, 2H), 8.06 (d,1H), 7.82 (s, 1H), 7.71 (d, 1H), 7.46 (d, 1H), 5.22 (t, 1H, CHCOO),3.45-3.75 (m, 2H, CH₂). MS (ES) m/e (M+1): 464. λ_(max) 328 nm, ε_(max)9,900 cm⁻¹M⁻¹ in MeOH.

B.

Methyl 6-(2-nitro-4-trifluoromethyl-phenoxy)quinolinyl-luciferin ester

The compound GST-4 was prepared by employing the similar method used forthe synthesis of MAO-12 using GST-3 as starting material.

¹H NMR (CD₂Cl₂): 8.23 (s, 1H), 8.0-8.2 (m, 3H), 7.75 (d, 1H), 7.50 (d,1H), 7.39 (s, 1H), 7.15 (d, 1H), 7.46 (d, 1H), 5.42 (t, 1H, CHCOO), 3.74(s, 3H, CH₃), 3.58 (d, 2H, CH₂). MS (ES) m/e (M+1): 478. λ_(max)(nm)/ε_(max) (cm⁻¹M⁻¹): 322/10,800; 328/8,800 in MeOH.

C.

6-(4-nitrophenoxy)quinolinyl-luciferin

The compound GST-5 was prepared by employing the similar method used forsynthesis of GST-3.

¹H NMR (d₆-DMSO): 8.39 (d, 1H), 8.28 (d, 2H), 8.19 (m, 2H), 7.74 (s,1H), 7.60 (d, 1H), 7.28 (s, 1H), 5.37 (t, 1H, CHCOO), 3.59 (d, 2H, CH₂).MS (ES) m/e (M+2): 397. λ_(max) 328 nm, ε_(max) 17,200 cm⁻¹M⁻¹ in MeOH.

D.

6-(2-nitrophenoxy)quinolinyl-luciferin

The compound GST-6 was prepared by employing the similar method used forthe synthesis of GST-3.

¹H NMR (d₆-DMSO): 8.39 (d, 1H), 8.13 (m, 3H), 7.78 (t, 1H), 7.63 (d,1H), 7.56 (s, 1H), 7.48 (t, 1H), 7.38 (d, 1H), 5.37 (t, 1H, CHCOO), 3.59(m, 2H, CH₂). MS (ES) m/e (M+2): 397. λ_(max) (nm)/ε_(max) (cm⁻¹M⁻¹):323/10,400; 327/9,500; 337/8,300 in MeOH.

E.

6-(3-trifluoromethyl-4-nitrophenoxy)quinolinyl-luciferin

The compound GST-7 was prepared by employing the similar method used forsynthesis of GST-3.

¹H NMR (d₆-DMSO): 8.47 (d, 1H), 8.15-8.30 (m, 3H), 7.85 (d, 1H), 7.78(d, 1H), 7.73 (dd, 1H), 7.55 (d, 1H), 5.45 (t, 1H, CHCOO), 3.5-3.7 (m,2H, CH₂). MS (ES) m/e (M+1): 464. λ_(max) 321 nm, ε_(max) 11,000 cm⁻¹M⁻¹in MeOH

F.

6-(2-trifluoromethyl-4-nitrophenoxy)quinolinyl-luciferin

The compound GST-8 was prepared by employing the similar method used forsynthesis of GST-3.

¹H NMR (d₆-DMSO): 8.56 (d, 1H), 8.47 (d, 2H), 8.20 (dd, 2H), 7.92 (d,1H), 7.72 (dd, 1H), 7.33 (d, 1H), 5.44 (t, 1H, CHCOO), 3.5-3.7 (m, 2H,CH₂). MS (ES) m/e (M+1): 464. λ_(max) 328 nm, ε_(max) 10,100 cm⁻¹M⁻¹ inMeOH.

G.

6-(5-trifluoromethyl-2-nitrophenoxy)quinolinyl-luciferin

The compound GST-9 was prepared by employing the similar method used forsynthesis of GST-3.

λ_(max) (nm)/ε_(max) (cm⁻¹M⁻¹): 321/10,400; 328/8,300 in MeOH

H.

6-(4-fluoro-2-nitrophenoxy)quinolinyl-luciferin

The compound GST-10 was prepared by employing the similar method usedfor synthesis of GST-3.

¹H NMR (d₆-DMSO): 8.38 (d, 1H), 8.02-8.2 (m, 3H), 7.6-7.8 (m, 2H),7.5-7.6 (m, 2H, 1H), 5.34 (t, 1H, CHCOO), 3.5-3.7 (m, 2H, SCH₂). MS (ES)m/e (M+2): 415. λ_(max) 321 nm, ε_(max) 10,600 cm⁻¹M⁻¹ in MeOH.

I.

6-(2-nitro-4-methylcarboxylphenoxy)quinolinyl-luciferin

The compound GST-11 was prepared by employing the similar method usedfor synthesis of GST-3.

¹H NMR (d₆-DMSO): 8.68 (d, 1H), 8.15-8.30 (m, 2H), 8.1-8.2 (m, 2H),7.64-7.74 (m, 2H), 7.33 (d, 1H), 5.42 (t, 1H, CHCOO), 3.81 (s, 3H, CH₃),3.5-3.7 (m, 2H, CH₂). MS (ES) m/e (M+2): 455. λ_(max) 321 nm, ε_(max)16,200 cm⁻¹M⁻¹ in MeOH

J.

6-(2-Nitro-benzenesulfonic acid)luciferin ester

Synthesis of 2-cyano-6-(2-nitro-benzenesulfonic acid)benzothiozole

To the solution of 6-hydroxy-2-cyanobenzothiozole (0.50 g, 2.84 mmol)and 2-nitrobenzene-sulfonyl chloride (0.63 g, 2.84 mmol) in 15 ml ofanhydrous methylene chloride was added TEA (0.58 g, 5.68 mmol). Theresultant mixture was stirred for 3 hours. The product was purified byflash chromatography using heptane/ethyl acetate/methylene chloride(70/30/15) as eluent in a yield of 55%.

Synthesis of GST-13

GST-13 was prepared by employing the similar method for the synthesis ofluciferin GST-3.

¹H NMR (d₆-DMSO): 8.14-8.26 (m, 2H), 8.17 (s, 1H), 8.07 (td, J=7.5 Hz,J=1.3 Hz, 1H), 7.99 (dd, J=8.0 Hz, Hz, J=1.2 Hz, 1H), 7.85 (td, J=7.8Hz, J=1.2 Hz, 1H), 7.34 (dd, J=9.0 Hz, J=2.4 Hz, 1H), 5.44 (t, J=9.0 Hz,1H, CH—COOH), 3.6-3.9 (m, 2H, CH₂). MS (ES): m/e (M+1), 466. λ_(max) 292nm, ε_(max) 19,100 cm⁻¹M⁻¹ in MeOH.

K.

6-(4-Nitro-benzenesulfonic acid)luciferin ester

The compound GST-14 was prepared by employing the similar method usedfor the synthesis of GST-13.

¹H NMR (d₆-DMSO): 8.42 (d, 2H), 8.17 (m, 3H), 8.08 (d, 1H), 7.25 (dd,1H), 5.44 (t, 1H, CH—COOH), 3.6-3.9 (m, 2H, CH₂). MS (ES): m/e (M+1),466. λ_(max) 292 nm, ε_(max) 19,400 cm⁻¹M⁻¹ in MeOH.

L.

6-(2-Nitro-benzenesulfonic acid)quinolinyl-luciferin ester

The compound GST-15 was prepared by employing the similar method usedfor the synthesis of GST-13.

¹H NMR (d₆-DMSO): 8.54 (d, 1H), 7.9-8.3 (m, 6H), 7.85 (t, 1H), 7.57 (dd,1H), 5.41 (t, 1H, CH—COOH), 3.5-3.7 (m, 2H, CH₂). MS (ES): m/e (M+1),460. λ_(max) 285 nm, ε_(max) 9,010 cm⁻¹M⁻¹ in MeOH.

M.

6-(4-Nitro-benzenesulfonic acid)quinolinyl-luciferin ester

The compound GST-16 was prepared by employing the similar method usedfor synthesis of GST-13.

¹H NMR (d6-DMSO): 8.23 (d, J=8.7 Hz, 1H), 8.43 (d, J=8.7 Hz, 2H), 8.18(m, 3H), 8.13 (d, J=9.3 Hz, 1H), 7.89 (d, J=2.7 Hz, 1H), 7.51 (dd, J=9.3Hz, J=3 Hz, 1H), 5.42 (dd, J=8.4 Hz, J=8.4 Hz, 1H, CHCOO), 3.5-3.7 (m,2H, CH₂). MS (ES) m/e (M+1): 461. λ_(max) 285 nm, ε_(max) 12,400 cm⁻¹M⁻¹in MeOH.

N.

6-(2-Nitro-4-trifluorobenzenesulfonic acid)luciferin ester

The compound GST-17 was prepared by employing the similar method usedfor synthesis of GST-13.

¹H NMR (d6-DMSO): 8.82 (s, 1H), 8.15-8.30 (m, 4H), 7.43 (d, 1H), 5.43(t, 1H, CHCOO), 3.6-3.9 (m, 2H, SCH₂). MS (ES) m/e (M+1): 534. λ_(max)292 nm, ε_(max) 18,600 cm⁻¹M⁻¹ in MeOH.

Other GST substrates are shown in FIG. 44B.

V. Fluorogenic GST/Glutathione Substrates

A.

7-(5-trifluoromethyl-2-nitrophenoxy)-4-methyl-coumarin

The compound was prepared by employing the similar method used forsynthesis of the precursor of GST-3.

¹H NMR (CD₂Cl₂): 8.37 (s, 1H), 7.88 (d, 2H), 7.70 (m, 2H), 7.28 (d, 1H),7.05 (d, 1H), 7.00 (s, 1H), 6.25 (s, 1H), 2.43 (s, 3H, CH₃). MS (ES) m/e(M+2): 367. λ_(max) 320 nm, ε_(max) 12,700 cm⁻¹M⁻¹ in MeOH

B.

Synthesis of bis(-(5-trifluoromethyl-2-nitrophenoxy)-fluorescein lactone

The mixture of fluorescein (2.0 g, 60 mmol),2-nitro-4-trifluoromethylbenzene chloride (3.0 g, 13.3 mmol) andpotassium carbonate (2.0 g, 14.5 mmol) in 50 ml of DMSO was heated to100° C. for 1 hour min. Upon cooling to room temperature, the mixturewas poured into 30 ml of cold water and exacted three times withmethylene chloride. The combined organic layer was washed with water anddried over magnesium sulfate. The product was purified by flashchromatography using heptane/methylene chloride/ethyl acetate (7/3/0 to7/3/) as eluent in a yield of 88%.

¹H NMR (CD₂Cl₂): 8.28 (s, br, 2H), 8.05 (d, 1H), 7.84 (d, 2H), 7.66-7.82(m, 2H), 7.26 (d, 2H), 7.03 (d, 2H), 6.92 (d, 2H), 6.84 (dd, 2H).

VI. Bioluminogenic Alkaline Phosphatase Substrates

A.

6-(4-phosphoric acid benzylether)luciferin

Synthesis of diethyl 4-(TBDMS-oxymethyl)phenyl phosphate

To the solution of 4-(TBDMS-oxymethyl)phenol (5.53 g, 23.2 mmol) and TEA(3.79 g, 37.4 mmol) was added diethyl phosphornyl chloride (4.31 g, 24.9mmol). The resultant mixture was stirred overnight. The compound waspurified with flash chromatography in a yield of 63%.

¹H NMR (CD₂Cl₂): 7.23 (d, 2H), 7.05 (d, 2H), 4.60 (s, 2H, OCH₂), 4.09(q, 4H, POCH₂), 1.15 (t, 6H, CH₃), 0.92 (s, 9H, CH₃), 0.12 (t, 6H,SiCH₃). MS (ES): m/e (M+2), 376.

Synthesis of diethyl 4-(hydroxymethyl)phenyl phosphate

The solution of HCl (8 ml) in ethanol (100 ml) was added to diethyl4-(TBDMS-oxymethyl)phenyl phosphate. The resultant solution was stirredfor 20 minutes and neutralized with sodium bicarbonate. The mixture wasextracted three times with ether and the combined organic layer wasdried over magnesium sulfate. After removal of solvent, the compound wasobtained in a yield of 98%. Without further purification, it wasdirectly used in next step.

¹H NMR (CD₂Cl₂): 7.38 (d, 2H), 7.10 (d, 2H), 4.65 (s, 2H, OCH₂), 4.09(q, 4H, POCH₂), 1.18 (t, 6H, CH₃). MS (ES): m/e (M+1), 261

Synthesis of diethyl 4-(bromomethyl)phenyl phosphate

To the solution of carbon tetrabromide (5.73 g, 17.3 mmol) and diethyl4-(hydroxymethyl)phenyl phosphate (3.74 g, 14.4 mmol) in 50 ml ofmethylene chloride was added triphenylphosphine (4.54 g, 17.3 mmol) at0° C. The resultant mixture was stirred for 2 hours. The compound waspurified by flash chromatography using heptane/ethyl acetate (80/20 to70/30) as eluent in a yield 92%.

¹H NMR (CD₂Cl₂): 7.41 (d, 2H), 7.20 (d, 2H), 4.53 (s, 2H, OCH₂), 4.20(q, 4H, POCH₂), 1.17 (t, 6H, CH₃). MS (ES): m/e (M+1), 323

Synthesis of phosphoric acid4-(2-cyano-benzothiazol-6-yloxymethyl)-phenyl diethyl ester

The compound was synthesized by employing the similar method for thesynthesis of precursor MAO-1.

¹H NMR (CD₂Cl₂): 8.12 (d, 1H), 7.49 (s, 1H), 7.46 (d, 2H), 7.33 (d, 1H),7.26 (d, 2H), 5.18 (s, 2H, OCH₂), 4.20 (m, 4H, POCH₂), 1.34 (t, 6H,CH₃). MS (ES): m/e (M+1), 420

Synthesis of 4-(2-cyano-benzothiazol-6-yloxymethyl)-phenyl phosphoricacid

To the solution of phosphoric acid4-(2-cyano-benzothiazol-6-yloxymethyl)-phenyl diethyl ester in 15 ml ofmethylene chloride was added iodotrimethylsilane (0.46 g, 2.31 mmol).The resultant mixture was stirred for 40 minutes and then added to thesolution of p-toluidine (1.50 g, 14.0 mmol) in 20 ml. After the solventwas partially removed, the light yellow precipitate was collected byfiltration and washed with ethanol and ether to give a yield of 63%.

¹H NMR (d6-DMSO): 8.18 (d, 1H), 8.0 (s, 1H), 7.43 (d, 2H), 7.38 (d, 1H),7.19 (d, 2H), 6.82 (d, 3.75H), 6.45 (d, 3.75H), 5.19 (s, 2H, OCH₂), 2.16(s, ˜6H, CH₃). (Containing about 3.3 molar equivalent toluidine). MS(ES): m/e (M+2), 362

Synthesis of AP-4

The compound was synthesized by employing the similar method used forthe preparation of MAO-1 and purified by HPLC using 5 mM ammoniumacetate buffer/acetonitrile as eluent.

¹H NMR (D₂O): 7.82 (d, 1H), 7.56 (s, 1H), 7.39 (d, 2H), 7.15 (d, 1H),7.10 (d, 2H), 5.11 (t, 1H, CH—COOH), 5.06 (s, 2H, OCH₂), 3.45-3.80 (m,2H, CH₂). MS (ES): m/e (M+2), 468. λ_(max) 329 nm, ε_(max) 19,100cm⁻¹M⁻¹ in water.

B.

6-Phosphoric acid-quinolinyl-luciferin

Synthesis of diethyl(2-cyano-quinolin-6-yl)phosphate

To the mixture of 2-cyano-6-hydroxyquinoline (0.50 g, 2.94 mmol) anddiethyl phosphorochloride (0.67 g, 3.53 mmol) in 15 ml of anhydrousmethylene chloride was added triethylamine (0.59 g, 5.88 mmol). Theresultant mixture was stirred for 5 hours at room temperature. Afterremoval of the solvent, the product was purified by flash chromatographyusing heptane/ethyl acetate as eluent in a yield of 91%.

¹H NMR (CD₂Cl₂): 8.35 (d, 1H), 8.18 (d, 1H), 7.80 (s, 1H), 7.6-7.8 (m,2H), 4.26 (m, 4H, CH₂), 1.18 (t, 6H, CH₃). MS (ES) m/e (M−2): 306.

Synthesis of (2-cyano-quinolin-6-yl) phosphoric acid

To the solution of diethyl (2-cyano-quinolin-6-yl) phosphate (0.58 g,1.86 mmol) in 10 ml of methylene chloride was added iodotrimethylsilane(0.82 g, 4.1 mmol). The resultant mixture was stirred for 40 minutes andthen added to the solution of p-toluidine (1.50 g, 14.0 mmol) in 20 ml.After the solvent was partially removed, the white yellow precipitatewas collected by filtration and washed with ethanol and ether to give ayield of 92%.

¹H NMR (d6-DMSO): 8.54 (d, 1H), 8.07 (d, 1H), 7.95 (d, 1H), 7.47 (d,1H), 7.82 (s, br, 1H), 7.71 (dd, 1H), 6.85 (d, 2.5H), 6.55 (d, 2.5H),2.13 (s, 3.85H, CH₃). (Containing 1.25 molar equivalent toludine ascounter-ions). MS (ES) m/e (M−2): 248

Synthesis of AP-2

The compound was synthesized by employing the similar method for thepreparation of AP-4.

¹H NMR (d6-DMSO): 8.29 (d, J=8.7 Hz, 1H), 8.05 (d, J=8.4 Hz, 1H), 7.95(d, J=9.0 Hz, 1H), 7.71 (d, J=2.4 Hz, 1H), 7.64 (dd, J=8.7 Hz, J=2.4 Hz,1H), 5.89 (dd, J=8.4 Hz, J=8.4 Hz, 1H, CHCOO), 3.4-3.65 (m, 2H, CH₂).(Containing ˜8 molar equivalent TEA/HCl as counter-ions). MS (ES) m/e(M+): 352

C.

Trimethyllock phosphate luciferin amide.

Synthesis of2-[3-(tert-Butyl-dimethyl-silanyloxy)-1,1-dimethyl-propyl]-3,5-dimethyl-phenol

To the solution of phenol (10.0 g, 31.2 mmol) and potassium t-butoxide(3.85 g, 34.3 mmol) in 30 ml of anhydrous THF was heated to 60° C. forminutes and then diethyl phosphate chloride (5.86 g, 33.96 mmol) wasadded. The resultant mixture was heated at 60-70° C. for 2.5 hours. Uponcooling to room temperature, the insoluble solid was removed byfiltration. The solvent of filtrate was removed and the residue waspurified by flash chromatography using heptane and ethyl acetate (90/10to 80/20) as eluent in a yield of 75%.

¹H NMR (CD₂Cl₂): 7.10 (s, 1H), 6.78 (s, 1H), 4.21 (q, 4H, OCH₂), 3.51(t, 2H, CH₂OSi), 2.58 (s, 3H, CH₃), 2.25 (s 3H, CH₃), 2.13 (t, 2H, CH₃),1.57 (s, 6H, CH₃), 1.37 (t, 6H, CH₃), 0.85 (s, 9H, CH3), 0.02 (s, 6H,SiCH₃). MS (ES): m/e (M+2), 460.

Synthesis of3-[2-(Diethoxy-phosphoryloxy)-4,6-dimethyl-phenyl]-3-methyl-butyric acid

To the solution of2-[3-(tert-Butyl-dimethyl-silanyloxy)-1,1-dimethyl-propyl]-3,5-dimethyl-phenolin 50 ml of acetone was added potassium fluoride (0.678 g, 11.6 mmol).The mixture was cooled at 0° C. and then Jone's reagent was added over30 minutes till the solution became deep orange. The resultant mixturewas allowed to warm up to room temperature and stirred for 3 hours. Theinsoluble solid was removed by filtration and washed several times withacetone. After removal of the solvent of the filtrate, the product waspurified by flash chromatography using heptane/ethyl acetate (70/30 to40/60) as eluent in a yield 87%.

¹H NMR (CD₂Cl₂): 7.02 (s, 1H), 6.79 (s, 1H), 4.25 (q, 4H, OCH₂), 2.90(s, 2H, CH₂), 2.52 (s, 3H, CH₃), 2.22 (s 3H, CH₃), 1.63 (s, 6H, CH₃),1.38 (t, 6H, CH₃). MS (ES): m/e (M+1), 359.

Synthesis of 2-cyano-6-(trimethyllock diethyl phosphate)benzothiazoleamide

To the solution of3-[2-(Diethoxy-phosphoryloxy)-4,6-dimethyl-phenyl]-3-methyl-butyric acid(0.91 g, 2.36 mmol) in 20 ml of anhydrous HF was addedisobutylchloroformate (0.36 g, 2.60 mmol) and N-methylmorpholine (0.26g, 2.60 mmol) at 0° C. The mixture was then stirred at room temperaturefor 30 minutes (the reaction should be checked by TLC to ensure acid iscompletely converted into anhydride). 6-Aminobenzothiazole (0.275 g,1.57 mmol) and N-methylmorpholine (0.53 g, 5.20 mmol) were added and themixture was stirred for 5 days at room temperature. After removal of thesolvent, the compound was purified by flash chromatography usingheptane/ethyl acetate (80/20 to 70/30) as eluent in a yield of 60%.

¹H NMR (CD₂Cl₂): 9.58 (s, 1H, NH), 8.55 (d, 1H), 7.98 (d, 1H), 7.38 (dd,1H), 7.0 (s, 1H), 6.69 (s, 1H), 4.36 (q, 4H, OCH2), 2.80 (s, 2H, CH₂),2.42 (s, 3H, CH₃), 2.20 (s, 3H, CH₃), 1.78 (s, 6H, CH₃), 1.48 (t, 6H,CH₃). MS (ES): m/e (M+1), 516.

Synthesis of 2-cyano-6-(trimethyllockphosphoric acid)benzothiazole amide

Diethyl phosphate trimethyllock amide was de-ethylated by employing thesimilar method used for the synthesis of AP-4 precursor.

¹H NMR (d6-DMSO): 8.62 (d, 1H), 8.03 (d, 1H), 7.50 (dd, 1H), 7.11 (s,1H), 6.98 (d, 4.5H), 6.75 (d, 4.5H), 6.48 (s, 1H), 2.86 (s, 2H, CH₂),2.38 (s, 3H, CH₃), 2.19 (s, ˜6.75H, CH3), 2.07 (s 3H, CH₃), 1.65 (s, 6H,CH₃). (Containing 2.25 equivalent molar toluididne as counter-ions) MS(ES): m/e (M−1), 459.

Synthesis of AP-5

The compound AP-5 was prepared by ring cyclization used for thesynthesis of AP-4 and purified by HPLC using 5 mM ammonium acetatebuffer/acetonitrile as solvent.

¹H NMR (D₂O): 7.80 (s, 1H), 7.77 (d, 1H), 7.21 (s, 1H), 7.06 (d, 1H),6.53 (s, 1H), 5.12 (t, 1H, CH—COOH), 3.5-3.8 (m, 2H, CH₂), 2.85 (s, 2H,CH₂), 2.28 (s, 3H, CH₃), 2.07 (s 3H, CH₃), 1.60 (s, 6H, CH₃). MS (ES):m/e (M+1), 564. λ_(max) 322 nm, ε_(max) 16,700 cm⁻¹M⁻¹ in water.

VII. Miscellaneous

A.

6-(4-phosphoric acid benzylether)luciferin

Synthesis of 4-formylphenol acetate

To the solution of 4-hydroxybenzaldehyde (10.0 g, 81.9 mmol) in 150 mlof pyridine was added acetic anhydride (60 ml). The resultant mixturewas stirred at room temperature for 2 hours. After removal of thesolvent, the compound was purified by flash chromatography usingheptane/methylene chloride/ethyl acetate (20/20/10) as eluent in a yieldof 93%.

¹H NMR (CD₂Cl₂): 10.0 (s, 1H, CHO), 7.86 (d, 2H), 7.30 (d, 2H), 2.32 (s,3H, CH₃)

Synthesis of 4-hydroxymethylphenol acetate

To the solution of 4-formylphenol acetate (6.0 g, 36.4 mmol) in 60 ml ofmethanol was added sodium boronhydride (1.50 g, 39.6 mmol) at 0° C. Theresultant mixture was stirred for 1 hour, neutralized with acetic acidand then poured into water. The mixture was extracted three times withether and the combined organic layer was dried over magnesium sulfate.After removal of solvent, the compound was purified by flashchromatography using heptane/ethyl acetate (80/20-70/30) as eluent in ayield of 41%.

¹H NMR (CD₂Cl₂): 7.4 (d, 2H), 7.08 (d, 2H), 4.67 (s, 2H, CH₂), 2.30 (s,3H, CH₃)

Synthesis of 4-bromomethylphenol acetate

To the solution of 4-hydroxymethylphenol acetate (2.50 g, 15.0 mmol) andcarbon tetrabromide (5.97 g, 18.0 mmol) in 40 ml of methylene chloridewas added triphenylphosphine (4.73 g, 180 mmol) under nitrogen. Theresultant mixture was stirred for 3 hours. The product was purified byflash chromatography using heptane/ethyl acetate (90/10-80/20) as eluentin a yield of 79%.

¹H NMR (CD₂Cl₂): 7.45 (d, 2H), 7.07 (d, 2H), 4.53 (s, 2H, CH₂), 2.29 (s,3H, CH₃)

Synthesis of 4-(2-cyano-benzothiazol-6-yloxymethyl)-phenol acetat

The compound was synthesized by employing the similar method for thesynthesis of precursor MAO-1.

¹H NMR (CD₂Cl₂): 8.14 (d, 1H), 7.49 (m, 3H), 7.26 (d, 1H), 7.14 (d, 1H),5.18 (s, 2H, OCH₂), 2.30 (s, 3H, CH₃).

Synthesis of E-1

The compound was synthesized by employing the similar method for thepreparation of GST-13.

¹H NMR (d6-DMSO): 8.05 (d, J=9.0 Hz, 1H), 7.86 (d, J=2.4 Hz, 1H), 7.51(d, J=8.7 Hz, 2H), 7.26 (dd, J=9.0 Hz, J=2.7 Hz, 1H), 7.15 (d, J=8.7 Hz,2H), 5.41 (dd, J=8.40 Hz, J=8.1 Hz, 1H, CH—COOH), 5.20 (s, 2H, OCH₂),3.6-3.80 (m, 2H, CH₂), 2.26 (s, 3H, CH₃). MS (ES): m/e (M+1), 429.λ_(max) 325 nm, ε_(max) 19,100 cm⁻¹M⁻¹ in MeOH

B.

6-(4-Chloro-phenylthiomethoxy)luciferin

Synthesis of 2-cyano-6-(4-chloro-phenylthiomethoxy)-benzothiazole

The mixture of 6-hydroxy-2-cyanobenzothiazole (0.45 g, 2.59 mmol),1-chloro-4-chloromethylthiobenzene (0.50 g, 2.59 mmol), potassiumcarbonate (0.36 g, 2.61 mmol) and sodium iodide (0.01 g) in acetone (30ml) was heated to reflux 30 minutes. Upon cooling to room temperature,the insoluble solid was removed by filtration. The solvent of filtratewas removed under reduced pressure, the residue was dissolved minimumamount of acetone with heating. The solution was cooled at 0° C. andyellow crystals were formed immediately. The solid was collected byfiltration to give a yield of 37%.

¹H NMR (CD₂Cl₂): 8.14 (d, 1H), 7.47 (d, 1H), 7.45 (d, 2H), 7.34 (d, 2H),7.32 (dd, 1H), 5.57 (z, 1H, CH₂).

Synthesis of 6-(4-chloro-phenylthiomethoxy) luciferin

The compound was made by employing the similar method for the synthesisof MAO-1 and was purified by flash chromatography.

¹H NMR (d6-DMSO): 8.06 (d, 1H), 7.87 (d, 1H), 7.47 (d, 2H), 7.20 (d,2H), 7.23 (dd, 1H), 5.79 (z, 1H, OCH₂), 5.22 (t, 1H, CH), 3.68 (d, 2H,CH₂). MS (ES): m/e (M+), 436. λ_(max) 324 nm, ε_(max) 16,300 cm⁻¹M⁻¹ inwater.

C.

6-(3,3-Dichloro-propoxy)-luciferin

Synthesis of 1,3-dichloropropanol

HCl gas was bubbled through acrolein (5.0 g, 89.2 mmol) at 0° C. for 3hours. To the reaction mixture was added methylene chloride (200 ml),and the resultant solution was washed with water three times and driedover magnesium sulfate. After removal solvent, the product was useddirectly in next step without further purification.

¹H NMR (CD₂Cl₂): 5.17 (t, 1H), 3.68 (t, 2H, OCH₂), 2.13 (q, 2H, CH₂).

Synthesis of 1,1,3-trichloropropane

To the mixture of PCl₅ (23.50 g, 0.113 mol) in 100 ml of petroleum ether(40-60° C.) was added 1,3-dichloropanol (14.5 g, 0.113 mol) dropwise.The solvent was evaporated at 40-60° C., the temperature was increasedto 120° C., and the fraction (70-90° C.) was collected under reducedpressure (70-80 mmHg). The distilled liquid was then poured intoice-water and stirred for 4 hours and extracted with petroleum ether(40-60° C.). The combined organic layer was dried over magnesiumsulfate. Repeat the above procedures until pH of the distilled liquid inaqueous solution is neutral. The final distillation gave a yield of 3.64g of product (22%).

¹H NMR (CD₂Cl₂): 6.03 (t, 1H), 3.74 (t, 2H, ClCH₂), 2.64 (q, 2H, CH₂).

Synthesis of 2-cyano-6-(3,3-Dichloro-propoxy)-benzothiazole

The compound was made by employing the similar procedure used for thesynthesis of MAO-1 precursor.

¹H NMR (CD₂Cl₂): 8.12 (d, 1H), 7.45 (d, 1H), 7.23 (dd, 1H), 6.51 (t, 1H,CHCl₂), 4.24 (t, 2H, OCH₂), 2.75 (q, 2H, SCH₂).

Synthesis of 6-(3,3-Dichloro-propoxy)-luciferin

The compound was made by employing the similar procedure used for thesynthesis of GST-3.

¹H NMR (d6-DMSO): 8.02 (d, 1H), 7.80 (d, 1H), 7.19 (dd, 1H), 6.46 (t,1H, CHCl₂), 5.35 (t, 1H, CHCOO), 4.24 (t, 2H, OCH₂), 3.6-3.8 (m, 2H,SCH₂), 2.69 (q, 2H, CH₂).

D.

Synthesis of (1-formyl-2-tritylthioethyl)carbamic acid tert-butyl ester

To the solution of t-BOC-trityl-Weinreb amide (1.67 g, 3.30 mmol) in 30of anhydrous ether was added LiAlH₄ (0.25 g, 6.59 mmol) in threeportions at −50° C. The temperature was allowed to warm up to −35° C.and the resultant mixture was stirred at −35° C. for 2 hours. Thereaction was quenched by adding 15 ml of ice-water slowly. The mixturewas extracted three times with ether and the combined organic layer wasdried over magnesium sulfate. After removal of the solvent, the productwas purified by flash chromatography using heptane/ethylacetate (8/2 to7/3) as eluent in a yield of 87%.

¹H NMR (CD₂Cl₂): 9.21 (s, 1H, CHO), 7.2-7.7 (m, 15H, Ar—H), 5.10 (s, br,1H, NHCO), 3.83 (m, 1H, CH), 2.4-2.8 (m, 2H, CH₂), 1.22 (s, 9H, CH₃).

Synthesis of (1-dimethylaminomethyl-2-trithioethyl)-carbamic acidtert-butyl ester

To the solution of triethylamine (0.11 g, 1.12 mmol) in absolute ethanol(5 ml) were added dimethylamine hydrochloride (0.0912 g, 1.12 mmol),titanium (IV) isopropoxide (0.317 g, 1.12 mmol) and(1-formyl-2-tritylthioethyl)carbamic acid tert-butyl ester (0.25 g, 0.56mmol). The reaction mixture was stirred for 10 hours at roomtemperature. NaBH₄ (0.045 g, 1.12 mmol) was added to the mixture, theresultant mixture was stirred for another 4 hours, and the reaction wasquenched by adding 10 ml of water the mixture. The mixture was extractedwith ether three times and the combined organic layer was dried overmagnesium sulfate. After removal of the solvent, the product waspurified by flash chromatography using methylene chloride/methanol aseluent (95/5) in a yield of 41%.

¹H NMR (CD₂Cl₂): 7.2-7.7 (m, 15H, Ar—H), 4.65 (s, br, 1H, NHCO), 3.62(m, 1H, CH), 2.42 (t, 2H, NCH₂), 2.20 (m, 2H, SCH₂), 2.17 (s, 6H, NCH₃),1.41 (s, 9H, CH₃). MS (ES): m/e (M+1), 477

Synthesis of dimethylaminoluciferin

To (1-dimethylamino-methyl-2-trithioethyl)-carbamic acid tert-butylester (0.18 g, 3.78 mmol) was added the solution of TFA (50%) andtriisopropylsilane (0.077 g). The resultant solution was stirred for 30minutes and 20 ml of ether was added. The solvent was removed underreduced pressure, the residue was triturated with ether and the yellowsolid was collected by filtration. Without further purification, thesolid was carried on next step.

The above solid and 2-cyano-6-hydroxybenzothiozole (0.067 g, 0.38 mmol)were dissolved in methanol (2.5 ml), CH₂Cl₂ (0.5 ml) and H₂O (1 ml). Tothe solution was added K₂CO₃ (0.052 g, 0.38 mmol). The mixture wasstirred at room temperature for 1 hour and then neutralized to slightlyacidic condition. After removal of organic solvent, the product waspurified by HPLC using 0.1% TFA water/acetonitrile as eluent.

¹H NMR (CD₃OD): 7.51 (d, 1H), 7.36 (d, 1H), 7.25 (dd, 1H), 6.46 (t, 1H,CHCl₂), 5.12 (m, 1H, CH), 3.75, 3.22 (m, 2H, SCH₂), 3.50 (m, 2H, NCH₂),3.04 (s, 6H, CH₃). MS (ES): m/e (M+), 293.

E.

Synthesis 6′-(2-hydroxyethoxyl) luciferin

The compound was synthesized by employing the similar method used forthe synthesis of GST-3.

¹H NMR (d6-DMSO): 8.0 (d, 1H), 7.72 (d, 1H), 7.17 (dd, 1H), 5.18 (t, 1H,CH), 4.08 (t, 2H, OCH₂), 3.74 (t, 2H, HOCH₂), 3.64 (m, 2H, SCH₂). MS(ES): m/e (M+1), 325. λ_(max) 328 nm, ε_(max) 16,500 cm⁻¹M⁻¹ in water.

F. 6′-(2,3,4,5,6-pentafluorobenzyloxy)-luciferin

6′(2,3,4,5,6-pentafluorobenzyloxy)-luciferin was synthesized in asimilar way as luciferin benzyl ether (LucBE) by using pentafluorobenzylbromide instead of benzy bromide as a starting material.6′(2,3,4,5,6-pentafluorobenzyloxy)-luciferin (compound of formula I′,III′, VII′, or VIII′) was tested in CYP assays.

G.6′-(2,3,4,6-tetrafluoro-5-((4-phenylpiperizin-1-yl)methyl)benzyloxy)-luciferin(LucPPXE4F)

This synthesis is same as the synthesis for6′-(3-((4-phenylpiperizin-1-yl)benzyloxy)-luciferin (LucPPXE) by usingα,α′-dibromo-m-tetrafluoroxylene, which was synthesized fromtetrafluoroisophthalic acid in two steps. The first step was reductionof tetrafluoroisophthalic acid with borane in THF to produceα,α′-dihydroxy-m-tetrafluoroxylene. The second step was to convert theα,α′-dihydroxy-m-tetrafluoroxylene intoα,α′-dibromo-m-tetrafluoroxylene. The major side product ismonobrominated compound.6′-(2,3,4,6-tetrafluoro-5-((4-phenylpiperizin-1-yl)methyl)benzyloxy)-luciferin(compound of formula IX′) was tested in CYP assays.

a. α,α′-dihydroxy-m-tetrafluoroxylene

To a solution of tetrafluoroisophthalic acid (2 g, 8.4 mmol) in 50 mL ofanhydrous tetrahydrofuran was added 1 M borane-tetrahydrofuran complexin tetrahydrofuran (34 mL, 33.6 mmol). The mixture was refluxed undernitrogen atmosphere for 4 hours. The solution was cooled down to roomtemperature, 200 mL of water was added and the resultant mixture wasextracted with ethyl ether (2×175 mL). The extracts were combined andwashed with saturated sodium bicarbonate (2×200 mL) and water (1×200mL). The organic phase was dried over anhydrous sodium sulfate and thesolvent was removed under reduced pressure. The residue was pumped overnight without further purification (1.3 g, 73.7% yield).

b. α,α′-dibromo-m-tetrafluoroxylene

To a mixture of lithium bromide (350 mg, 4 mmol) in 5 mL of anhydrousacetonitrile was added trimethylsilyl chloride (650 μL, 5 mmol). To thissolution was the added α,α′-dihydroxy-m-tetrafluoroxylene (210 mg, 1mmol) in 10 mL of anhydrous acetonitrile. The resultant mixture was thenrefluxed under nitrogen atmosphere for 6 hours. The mixture was thentaken up by 40 mL of ethyl ether, washed successively with water (2×40mL), 1M sodium bicarbonate (1×40 mL), and brine (1×40 mL). The organicphase was then dried over sodium sulfate and the solvent was removedunder reduced pressure and the residue was then purified by flashchromatography with 70% heptane in ethyl acetate (85 mg, 25% yield).

B. Synthetic Schemes for Derivatives for Redox or DealkylaseBioluminescent Assays

The appropriately substituted ortho-aminohalopyridine is converted tothe 2-thiohydroxylpyridothiazole followed by alkylation with methyliodide to yield a 2-methylthiopyridothiazole. The thioetherfunctionality is oxidized to the sulfone. The pyridine amine is eitheroxidized to the N-oxide or methylated depending on the desired product.The sulfone is displaced with cyanide to yield the corresponding2-cyanopyridothiazole. Cysteine (D, L, DL with or without the carboxylicacid esterified) is condensed with the cyanopyridothiazole. Anyremaining protecting groups are removed by standard means known in theart to yield the appropriate methylated or oxidized aza-luciferins.

Examples of Starting Orthoaminohalopyridines

References for Synthesis of Either Example Starting Materials

Starting materials 1, 3 and 4 are commercially available. Startingmaterial 2 is described in Mazeas, Heterocycles, 50:1065 (1999)),starting material 5 in Kuramochi (U.S. published application20050004103); and starting material 6 in Kolesnikov (U.S. publishedapplication 20030225036).

The N3-oxide of luciferin is synthesized by oxidation of6-hydroxy-2-methylbenzthiazole or 6-amino-2-methylbenzthiazole to yieldthe oxidized methylbenzthiazole. The methyl is converted to a nitrileusing procedures similar to those known in the art (Takahashi et al.,Chem & Pharm. Bull., 18:1176 (1970); Sasson et al., Org Lett., 7:2177(2005))). The nitrile is condensed with cysteine (D, L, DL, with orwithout the carboxylic acid esterified).

The N1-oxide of quinolinyl luciferin is synthesized by oxidation of2-methyl-6-hydroxyquinoline or to yield the oxidized methylquinolinol.The methyl is converted to a nitrile using procedures similar to thoseknown in the art (Takahashi et al., Chem & Pharm. Bull., 18:1176 (1970);Sasson et al., Org Lett., 7:2177 (2005)). The nitrile is condensed withcysteine (D, L, DL, with or without the carboxylic acid esterified).

The invention will be described by the following non-limiting examples.

Example 1 Measurement of Glutathione S Transferase Activity orGlutathione with Luciferin Derivatives

A. Assay at pH 6.6

A luciferin derivative, GST #3, was prepared as a substrate for GST. Thederivative was tested in a two step format. For the first step, thederivative was added to a mixture with one of three GSTs with or withoutglutathione. At different times after the reaction was initiated, aportion was removed and mixed with a luciferase reaction mixture.Reactions in which light production increased over time indicate thatGST#3 is a substrate for the GST in that reaction.

Materials and Methods

Equine GST Solution: 25 mg of equine GST (Part G 6511, Sigma ChemicalCompany, St. Louis, Mo.) was dissolved in 5 ml of 10 mM BisTris buffer,pH 6.6. Porcine GST Solution: 10 mg of porcine GST (Sigma ChemicalCompany G 6636) was dissolved in 2 ml of 10 mM BisTris, pH 6.6. S.japonica GST: 1 vial with 3 mg GST (Part G5663, Sigma Chemical Company)was dissolved in 500 μl of 10 mM BisTris, pH 6.6. Glutathione (reduced)(Part G 4251, Sigma Chemical Company) was dissolved in water to create a100 mM solution. GST #3 was dissolved in acetonitrile to produce a 12 mMsolution.

A bottle of Luciferin Detection Reagent (V859B, Promega Corp., Madison,Wis.) was dissolved with a bottle of P450-Glo Buffer (V865B, PromegaCorp.), thawed and allowed to come to room temperature. A 10 ml sampleof the resulting solution was mixed with 8 ml of water to create aluciferin detection solution.

The reactions (Table 1) were assembled by first adding the water and 1 MBisTris solutions in individual 0.5 ml microfuge tubes, then adding theother components except for the GST #3 solution. Finally, the GST #3(FIG. 12) solution was added to the reactions, the resulting solutionwas mixed, and the time of addition noted. At 0.1, 5, 10, and 15 minutespost mixing, a 10 μl sample was mixed with 90 μl of the luciferindetection solution and the light produced by the solution wasimmediately read using a Turner TD 20/20 Luminometer (Promega Corp.).

TABLE 1 Reaction Component 1 2 3 4 5 6 7 8 GST substrate 5 μl 5 μl 5 μl5 μl 5 μl 5 μl 5 μl 5 μl 1 M BisTris pH 6.6 5 μl 5 μl 5 μl 5 μl 5 μl 5μl 5 μl 5 μl 100 mM Glutathione 2 μl 2 μl 2 μl 2 μl (red) RedissolvedS.j. GST 5 μl 5 μl Redissolved porcine 5 μl 5 μl GST Redissolved equineGST 5 μl 5 μl Water 90 μl  88 μl  85 μl  83 μl  85 μl  83 μl  85 μl  83μl Results

Reactions were assembled with a luciferin derivative, a glutathione Stransferase (GST) from one of three sources, in the presence and absenceof glutathione. Samples from those reactions were then added to aluciferase reaction mixture. The light production from the mixture ofthe reactions with the luciferin detection reagent was measured using aTurner TD 20/20 Luminometer. The light values measured for the reactionsare shown in Table 2.

TABLE 2 Time (minutes) 1 2 3 4 5 6 7 8 0.1 33.26 35.72 32.67 32.57 46.9571.1 30.42 252.2 5 38.2 36.07 32.42 40.79 51.85 269.1 32.98 3662 1032.17 40.85 38.65 48.64 51.15 463.2 38.75 7629 15 35.53 46.43 33.8261.54 48.41 619.3 38.39 9324

The light values obtained from reactions without glutathione even in thepresence of GST (reactions 1, 2, 3, 5 and 7) did not increase greatlyover time. However, the light values measured in reactions containingboth glutathione and GST in addition to GST #3 increased substantiallyover time. That result indicated that the action of the transferase incombination with glutathione results in production of a product thatallows increased light production from the luciferase based reactionrelative to the starting solution, and that the luciferin derivativepermitted detection of the GST.

B. Assay at Slightly Basic pH

Common assays for GST are performed at a pH value around pH 6.5 eventhough the enzyme does not display maximal activity at this pH. Theselection of such a suboptimal pH is the result of the need to preventthe rapid, non-enzymatic attack of glutathione itself on the substrate,which leads to a very high background value for control reactions run tomeasure this rate. To determine whether a luciferin derivative that is aGST substrate was useful in reactions at lower pHs, the luciferinderivative, one of the GSTs and a reagent (glutathione) from Example 1Awere tested over pHs from 6.5 to 8.5.

Materials and Methods

Buffer stock solutions (1 M) of BisTris at pH 6.5, HEPES, pH 7.0, 7.5and 8.0 and Tricine pH 8.5 were prepared by dissolving solid reagentobtained from Sigma Chemical Corporation and adjusting the pH to thedesired value. These new buffer stocks were used with stock reagents inExample 1A to produce the solutions shown in Table 3.

TABLE 3 100 mM Reaction Buffer GST #3 Water Glutathione 1 5 μl of 1 MBisTris 2 μl 93 μl 0 pH 6.5 2 5 μl of 1 M BisTris 2 μl 88 μl 5 μl pH 6.53 5 μl of 1 M HEPES, 2 μl 93 μl 0 pH 7.0 4 5 μl of 1 M HEPES, 2 μl 88 μl5 μl pH 7.0 5 5 μl of 1 M HEPES, 2 μl 93 μl 0 pH 7.5 6 5 μl of 1 MHEPES, 2 μl 88 μl 5 μl pH 7.5 7 5 μl of 1 M HEPES, 2 μl 93 μl 0 pH 8.0 85 μl of 1 M HEPES, 2 μl 88 μl 5 μl pH 8.0 9 5 μl of 1 M Tricine, 2 μl 93μl 0 pH 8.5 10 5 μl of 1 M Tricine, 2 μl 88 μl 5 μl pH 8.5

The GST #3 addition was performed last and the solutions were mixedimmediately after addition of GST #3. After mixing, 10 μl samples wereremoved immediately and at 20 and 40 minutes post mixing, and thesamples were added to 90 μl of luciferin detection solution assembled asdescribed in Example 1A. After mixing, the light produced by thesolution was measured using a Turner TD 20/20 luminometer.

Results

Reactions were assembled at various pH values and the rate oftransformation of GST #3 was measured in the presence and absence ofglutathione. Reactions were then assembled containing S. japonica GSTwith and without glutathione. The light values were recorded (Table 4).

TABLE 4 Light Output time of 20 minutes post 40 minutes post Reactionmixing mixing mixing 1 1.01 0.976 1.099 2 1.13 1.287 1.761 3 1.198 1.3681.202 4 1.156 1.178 2.2 5 1.19 1.214 1.194 6 1.171 2.788 5.173 7 1.1671.172 1.236 8 1.198 6.662 13.38 9 1.182 1.199 1.246 10 1.342 25.24 47.01

The light signal from the incubations containing glutathione (reactions2, 4, 6, 8, and 10) increased over time while those from the incubationswithout glutathione did not change greatly. In fact, the rate of signalincrease was greater as the pH of the solution was varied from pH 6.5 to8.5. This rise in light value could be used as a way to measureglutathione in a solution, particularly if the solution is incubatedwith an appropriate luciferin derivative at an elevated pH. As shown inExample 1, this particular GST did not produce a very strong lightsignal when used at a relatively high enzyme concentration. However, byrunning the GST reaction at an elevated pH, a lower amount of thisenzyme was shown to generate a stronger signal.

The signal produced from reactions containing GST were compared withthose measured in the absence of GST to determine if the net signal seenwith enzyme becomes very small versus that produced by the enzyme. Inaddition, the stock of 100 mM glutathione (GSH) was diluted 1:10 withwater to produce a 10 mM GSH stock and this solution was added atvarious levels to determine if a lower non-enzymatic light signal couldbe produced while maintaining the same net light increase upon enzymeaddition.

The stock of S. japonica GST used in Example 1A was diluted to 1 mg/mlwith 10 mM BisTris pH 6.5. The solutions which were assembled are shownin Table 5.

TABLE 5 GST Amt GSH Rx #3 Buffer buffer Enzyme (10 mM) Water 1 5 μl 1 MHEPES, pH 7.5 5 μl 0 50 μl 40 μl 2 5 μl 1 M HEPES, pH 7.5 5 μl 10 μl 50μl 30 μl 3 5 μl 1 M HEPES, pH 7.5 5 μl 0 30 μl 60 μl 4 5 μl 1 M HEPES,pH 7.5 5 μl 10 μl 30 μl 50 μl 5 5 μl 1 M HEPES, pH 7.5 5 μl 0 10 μl 80μl 6 5 μl 1 M HEPES, pH 7.5 5 μl 10 μl 10 μl 70 μl 7 5 μl 1 M HEPES, pH8.0 5 μl 0 50 μl 40 μl 8 5 μl 1 M HEPES, pH 8.0 5 μl 10 μl 50 μl 30 μl 95 μl 1 M HEPES, pH 8.0 5 μl 0 30 μl 60 μl 10 5 μl 1 M HEPES, pH 8.0 5 μl10 μl 30 μl 50 μl 11 5 μl 1 M HEPES, pH 8.0 5 μl 0 10 μl 80 μl 12 5 μl 1M HEPES, pH 8.0 5 μl 10 μl 10 μl 70 μl 13 5 μl 1 M Tricine, pH 8.5 5 μl0 50 μl 40 μl 14 5 μl 1 M Tricine, pH 8.5 5 μl 10 μl 50 μl 30 μl 15 5 μl1 M Tricine, pH 8.5 5 μl 0 30 μl 60 μl 16 5 μl 1 M Tricine, pH 8.5 5 μl10 μl 30 μl 50 μl 17 5 μl 1 M Tricine, pH 8.5 5 μl 0 10 μl 80 μl 18 5 μl1 M Tricine, pH 8.5 5 μl 10 μl 10 μl 70 μl

The reactions were initiated by addition of GST #3 and mixing. At 45minutes post mixing, 10 μl samples were removed and added to 90 μl ofthe luciferin detection reagent and the light read using a Turner TD20/20 Luminometer. These readings are shown in Table 6.

TABLE 6 Light Reaction Reading 1 26.3 2 61.58 3 19.21 4 55.18 5 10.73 652.23 7 65.98 8 152.3 9 48.41 10 135.6 11 22.52 12 97.47 13 295.2 14532.4 15 199.4 16 416.7 17 97.89 18 267.3

The data in Table 6 can be rearranged to allow easy calculation of thesignal change with and without enzyme at the pH levels and glutathionelevels used (see Table 7).

TABLE 7 Reaction Reaction Net light Reaction pH with enzyme w/o enzymedifference 7.5 5 mM GSH 26.3 61.58 35.28 3 mM GSH 19.21 55.18 35.97 1 mMGSH 10.73 52.23 41.5 8.0 5 mM GSH 65.98 152.3 86.32 3 mM GSH 48.41 135.687.19 1 mM GSH 22.52 97.47 74.95 8.5 5 mM GSH 295.2 532.4 237.2 3 mM GSH199.4 416.7 217.3 1 mM GSH 97.89 267.3 169.41

These results confirm that GST #3 can be used to measure glutathione bymeasuring the light produced in reactions not containing GST. Inaddition, they indicate that stronger net light signals can be seen inreactions at pH values above pH 7.5. Finally, the results indicate thatlowering the glutathione level from 5 mM to a lower level resulted in alower light signal and that the net signal produced by S. japonica GSTdid not change greatly when the glutathione level was reduced from 5 mMto 3 mM.

C. Detection and Measurement of Glutathione

In Example 1B, it was shown that a luciferin derivative can betransformed to a species that produces a more intense light signal whenthe added solution contains glutathione. Moreover, in reactions that didnot contain enzyme, the results indicated that a non-enzymatictransformation was possible. To detect much lower levels of glutathione,the ability of GST to catalyze this conversion was utilized in thepresence of much lower levels of glutathione.

Materials and Methods

Master mix: 200 mM HEPES buffer, pH 7.5 also containing 0.4 mg/ml blotqualified BSA (Promega Corp. W3841), and 200 μg/ml equine GST (dilutedfrom a 5 mg/ml solution of equine GST, Sigma Chemical Corp. enzyme).Substrate (GST #3): 200 μM GST #3 in water. DTT: A stock of 20 mM DTTwas made by dissolving solid DTT (Promega Corp, V3151) in water. Thisstock was used to create a 4 mM DTT stock by dilution with water. 2Mercaptoethanol: A 20 mM solution of 2 mercaptoethanol was made bydiluting neat 2 mercaptoethanol (M6250, Sigma Chemical Corp.) in water.This stock was used to create a 4 mM 2-mercaptoethanol stock by dilutionwith water. A 100 mM glutathione stock (reduced) was diluted to producesolutions at 400, 320, 240, 200, 160, 80, and 20 μM glutathione bydilution into distilled water.

Cell Extract Samples: A solution of diluted rabbit reticulocyte lysatewas prepared by diluting a rabbit reticulocyte lysate (Promega Corp.)with water at a ratio of 2 μl lysate per 25 μl of solution. A solutionof diluted wheat germ lysate was made by diluting wheat germ extract(Promega Corp.) with water at a ratio of 2 μL extract per 25 μl ofsolution.

Luciferin Detection Reagent (see Example 1A).

The reactions were performed at room temperature in a microtiter plate.The reactions were initiated by the addition of the Master Mix solution.The following materials were added to the wells in the microtiter plate.

Columns 1-3: 25 μl of substrate solution and 25 μl of water were addedto all wells in columns 1-3 rows A-H. In addition, 25 μl of glutathionesolution at 400, 320, 240, 260 80 and 20 mM, was added to rows A-G,respectively. An additional 25 μl of water was added to columns 1-3 rowH.

Columns 4-6: 25 μl of substrate solution and 25 μl water were added tocolumns 4-6 rows A, C, E and G. Additions of 25 μl substrate and 25 μlof 200 μM glutathione were added to columns 4-6 rows B, D, F and H.Then, 25 μl of 4 mM DTT was added to columns 4-6 rows A and B; 25 μl of20 mM DTT was added to columns 4-6 rows C and D; 25 μl of 4 mM 2mercaptoethanol was added to columns 4-6 rows E and F, and 25 μl of 20mM 2 mercaptoethanol was added to columns 4-6 rows G and H.

Columns 7-9. These reactions were assembled as for columns 1-3 with theexception that the 25 μl of water was replaced with 25 μl of dilutedwheat germ extract.

Columns 10-13. These reactions were assembled as for columns 1-3 withthe exception that the 25 μl of water was replaced with 25 μl of dilutedrabbit reticulocyte lysate.

After all these solutions were assembled, the reactions were started byaddition of 25 μl of master mix to the solutions using a multichannelpipette and mixing the solution by pipetting up and down 3 times.Immediately after mixing, 10 μl of the solutions were removed with amultichannel pipette and added to 90 μl of Luciferin Detection Reagentin a Microtiter Luminometer Plate. After addition of samples from allwells of the original microtiter plate, the luminometer plate was sealedwith a clear plate sealer, allowed to sit at room temperature for 15minutes and then the light produced was read on a VERITAS™ luminometer.

Additional 10 μl samples were taken from the original microtiter plate,diluted into Luciferin Detection Reagent and read as above at 14.8,33.7, 44.75, 59.6 and 140 minutes post addition of master mix.

Results

Reactions with GST#3, with or without GST, and with or without a sourceof glutathione were prepared, and at various times after initiation ofthe reactions, a portion was added to a luciferase reaction mixture. Thelight readings from the triplicate reactions were averaged and are shownin the tables below.

TABLE 8 Light Signals Measured in Reactions Without Added Lysate orReductants Glutathione Concentration (μM) Time Added (minutes) 0 5 10 2040 60 80 100 0 1234 1592 2269 2821 5110 5248 8038 6604 14.8 2689 4310773802 125110 218325 273665 324305 337276 33.7 4362 90387 154525 277975444877 552806 693907 773268 44.75 5378 107668 195083 361281 567948741797 888763 935822 58.6 6015 142988 264698 694628 702468 956594 9924551197938 140 11744 280230 498136 830155 1268954 1796833 2129202 2480280

The data in Table 8 demonstrate that even a solution of 5 μM glutathionecan produce a light signal far above that seen in the absence of anyglutathione and that the signal increases over time and over glutathioneconcentration at least over the ranges of values tested.

TABLE 9 Light Signals Measured in Reactions With Wheat Germ ExtractGlutathione Concentration Added Time (μM) (minutes) 0 5 10 20 40 60 80100 0 1851 1964 2390 2512 4371 3611 5335 4235 14.8 49004 71860 90718115137 136851 182746 203690 218906 33.7 99432 152066 189073 279159330741 435730 504985 519121 44.75 138755 195962 237008 333459 480389540647 627461 558359 58.6 179435 244615 289289 618643 569417 586894715805 835949 140 362693 465865 511594 816161 1053306 1316857 12651441809120

The light values measured in the reactions containing wheat germ extract(Table 9) without added glutathione increased much more rapidly than isseen for reactions that did not contain added glutathione or wheat germextract (compare the 0 μM glutathione addition columns in the tablesabove). However, the presence of the extract also produced a lower netlight signal in reactions given a glutathione spike above about 30 μM(see Table 10).

TABLE 10 Net Light Signal in Reactions With or Without Wheat GermExtract Glutathione Concentration Added Time (μM) (minutes) 0 5 10 20 4060 80 100 0 617 372 120 −309 −739 −1637 −2703 −2369 14.8 46316 2875316916 −9973 −81473 −90918 −120614 −118371 33.7 95071 61679 34548 1185−114136 −117076 −188922 −254147 44.75 133377 88294 41926 −27822 −87559−201150 −261302 −377463 58.6 173420 101627 24591 −75985 −133051 −369700−276650 −361989 140 350949 185634 13458 −13994 −215648 −479975 −864057−671160

Thus, the wheat germ extract most likely contains a significant level ofglutathione and also may contain an interfering substance that reducedthe level of signal generated at high glutathione concentrations.However, one technique that is used to estimate the level of startingcompound in a sample which may have both a significant level of analyteyet also have an interfering substance is to determine if the signalincreases in a relatively linear manner with added analyte and to thendetermine the amount of added analyte needed to produce a signal in thesample that is twice the signal generated in the sample without addedanalyte. In this case, the signal increase was relatively linear withadded analyte to the sample and thus the value of glutathione in thediluted extract can be estimated to produce a glutathione concentrationin the reaction of between 10 and 20 μM glutathione. Since this increasewas seen from a 2 μl sample of starting extract, the estimatedconcentration in the actual sample was between 500 μM and 1 mMglutathione.

The light readings in reactions containing rabbit reticulocyte lysatebut without added glutathione increased much more rapidly over reactionswithout added glutathione or lysate (compare readings over time in Table8, no glutathione added column to the no glutathione added column inTable 11).

TABLE 11 Light Signals Measured in Reactions Including RabbitReticulocyte Lysate Glutathione Concentration Added Time (μM) [min] 0 510 20 40 60 80 100 0 2084 1816 1890 2168 2876 2754 3937 2822 14.8 8340387258 90540 104181 130163 144804 152024 164924 33.7 171939 176106 188794229026 269989 312938 348543 378781 44.75 229277 232897 237699 290097345094 386012 448978 453140 58.6 292086 306830 308048 445066 442405504909 553766 587558 140 572193 572518 610619 705215 819448 9810571097061 1184652However, with the rabbit reticulocyte lysate, a much smaller net signalincrease was seen when glutathione was spiked into the lysates and thesignal did not double until 100 μM glutathione was added in the spike(Table 11). The signal increases produced by spiking glutathione intothe reactions was again approximately linear with glutathione added andthus again allowed the glutathione concentration present from the sampleto be estimated. However, in this case, the estimated finalconcentration of glutathione generated by dilution of the lysate itselfis 100 μm. Since this increase was seen from a 2 μl sample of thestarting extract, the estimated concentration in the original lysate isapproximated to be between 2.5 and 5 mM glutathione.

The averaged results from the reactions performed with DTT or2-mercaptoethanol with or without spiked glutathione are presented inTable 12.

TABLE 12 Average Signals in Reactions with Added DTT or2-mercaptoethanol (ME) 50 μM Time (minutes) Reductant glutathione 0 14.833.7 44.75 58.6 140 none none 1234 2689 4362 5378 6015 11744 1 mM DTT −1672 61526 145772 153924 220284 565135 1 mM DTT + 7220 305825 731311857849 1069468 1944007 5 mM DTT − 2332 67536 155069 197547 241226 5265015 mM DTT + 7408 266152 636054 791397 960823 1648897 1 mM 2-ME − 112324736 62528 90732 S.L.* 260865 1 mM 2-ME + 5209 259259 542606 626141S.L. 1763802 5 mM 2-ME − 1442 34787 88417 118636 160571 339791 5 mM2-ME + 5708 252675 606326 850061 1060698 2073466 none +** 5179 245995498842 654873 829531 1532893 *samples lost, **estimated from results of40 and 60 μM glutathione reactions presented in Table 11.

The reactions with reducing agent but without added glutathione show alight signal well above that seen without these reducing agents.However, when the net signal is calculated from these results, the netsignals in the presence of these reductants was very near that seen intheir absence (Table 13).

The reaction scheme was relatively insensitive to the presence of otherreducing agents in the sample and it can be used in at least two complexcell lysates, wheat germ extract and rabbit reticulocyte lysate.

TABLE 13 Net signal in Reactions With and Without Reductant Time(minutes) Reductant 0 14.8 33.7 44.75 58.6 140 None 3945 243306 494480649495 823516 1521149 1 mM DTT 5548 244299 585540 703926 849184 13788725 mM DTT 5077 198616 480985 593850 719597 1122396 1 mM 2-ME 4086 234523480078 535409 N.A. 1502937 5 mM 2-ME 4266 217888 517909 731426 9001271733675D. Use of Luciferin Esters for Measurement of Glutathione and GST

To determine whether different GSTs had a preference for certainluciferin derivatives, a bioluminogenic assay was conducted with variousGSTs (or a control with glutathione alone) and acetyl luciferin.

Materials and Methods

Enzyme and glutathione stock solutions are described in Example 1A.

Acetyl luciferin was dissolved at 9 mM in acetonitrile.

P450 Glo Reagent was made by dissolving a bottle of Luciferin DetectionReagent (V859B, Promega Corp.) with a bottle of P450-Glo Buffer (V865B,Promega Corp.)

Thirty-six 90 μl reaction solutions were created that were designed tocontain the following materials when diluted to 100 μl: reactions 1-3,50 mM HEPES pH 7.5, 20 μM acetyl luciferin; reactions 4-18, 50 mM HEPESpH 7.5, 20 μM acetyl luciferin, 2 mM glutathione; reactions 19-21, 50 mMHEPES, pH 7.5, 2 μM acetyl luciferin, and; reactions 22-36, 50 mM HEPES,pH 7.5, 2 μM acetyl luciferin, 2 mM glutathione. After assembly, theenzyme stock solutions were diluted 1:10 into 10 mM HEPES, pH 7.5 andthe following additions were made to the reactions.

GST A1-1 (Sigma) is an enzyme found in a few body tissues, includingkidney, intestine, lung and liver. GST M1-1 (Sigma) is a type b allelicvariant, which corresponds to GST psi purified from human liver.

Reaction Reaction Type Addition 1-3 and 19-21 Buffer control 10 μl water4-6 and 22-24 Glutathione alone 10 μl water 7-9 and 25-27 S. japonicaGST 10 μl 1:10 S. japonica GST 10-12 and 28-30 Equine GST 10 μl 1:10Equine GST 13-15 and 31-33 GST A1-1 10 μl 1:10 GST A1-1 16-18 and 34-36GST M1-1 10 μl 1:10 GST M1-1

The reactions were allowed to proceed for 30 minutes at room temperatureand then 100 μl of P450 Glo reagent was added to each reaction and thelight produced by the subsequent solution was measured on a VERITAS™luminometer. The readings recorded are shown in Table 14.

TABLE 14 Raw Data Net Enzyme 20 mM Glutathione Signal Reaction AL* 2 mMAL Signal Net 20 mM Net 2 mM Buffer Alone 183,852 20,649 GlutathioneAlone 330,916 35,206 147,064 14,557 S. japonica GST 1,233,119 126,288902,203 91,082 Equine GST 361,356 39,036 30,440 3,830 GST A1-1 365,77042,725 34,854 7,519 GST M1-1 419,400 42,867 88,484 7,661 *AcetylLuciferinResults

The results above demonstrate that a substantial non-enzymatic signalcan be generated by incubation of acetyl luciferin with glutathione.Thus, such incubations can be used to detect and measure the presence ofglutathione in a sample.

In addition, the results indicated that a much stronger net signal wasgenerated by S. japonica GST with acetyl luciferin relative to the otherGST enzymes. This is in contrast to the results seen with GST #3, whereS. japonica GST did not generate nearly as strong of a net signal thanis seen with the other enzymes (Example 1). Thus, modification of thechemical groups attached to luciferin can change the relative preferenceof the resulting substrate for various GST enzymes.

In this example, an ester of luciferin (acetylluciferin) wasdemonstrated to be utilized by various GST enzymes but with morereactivity towards the enzyme from S. japonica than for mammalian GSTenzymes, the reverse of the preference seen with GST #3. Acetylluciferin was also shown to be able to react in solutions with 2 mMglutathione

Summary

In a manner similar to that used in Example 1A, other luciferinderivatives were shown to be substrates with various preferences forGSTs (see FIG. 12). Thus, luciferins modified in various ways can betransformed by GST in the presence of glutathione to a form that can beutilized more effectively by luciferase, as demonstrated by anincreasing light signal. Moreover, glutathione concentrations can bemeasured at high concentration through the direct attack of this agentonto the luciferin derivative or at much reduced concentration through areaction catalyzed by GST. Further, GST enzymes show differentialpreferences for luciferin derivative, dependent upon the particularmodifications present on the luciferin derivative.

In particular, GST#3 and a sample suspected of having a GST may beassayed at pHs from 6.5 to at least 8.0. The ester of GST#3 was a bettersubstrate for GST M1-1 in a light generating reaction than GST A1-1,while GST#3 was a better substrate for equine GST in a light generatingreaction than GST M1-1 and GST A1-1.

For some derivatives, a detergent may be added to the reaction mixtureto enhance the solubility of the derivative. For example, a detergentsuch as Triton X-114, e.g., about 0.01 to about 20%, including about 0.1to about 0.5% Triton X-114, was added to reaction mixtures havingGST-22.

Example 2 A. Detection of Equine Alcohol Dehydrogenase

A luciferin derivative, luciferol, was employed as a substrate foralcohol dehydrogenase (ADH). The derivative was tested in a two stepformat. For the first step, the derivative was added to a mixture withone of four ADHs. At different times after the reaction was initiated, aportion was removed and mixed with a luciferase reaction mixture.Reactions in which light production increased over time indicate thatluciferol is a substrate for the ADH in that reaction.

Materials and Methods

A solution of 500 mM Bis Tris, pH 6.5 was created by dissolving thesolid and adjusting the pH to pH 6.5. A 50 mM solution was then madefrom this stock by dilution with water.

Al DH solution: 25 units of aldehyde dehydrogenase (Sigma Chemical Co,A-6338) was dissolved in 1 ml of 50 mM Bis Tris, pH 6.5.

Eq ADH solution: 20 units of equine alcohol dehydrogenase (Sigma Chem.Co., A9589) was dissolved in 1 ml of 50 mM Bis Tris, pH 6.5.

Y ADH solution: 7500 units of yeast alcohol dehydrogenase (Sigma Chem.Co. A 7011) was dissolved in 1 ml of 50 mM Bis Tris pH 6.5.

NAD+ solution: 66 mg of NAD+ (Sigma Chem. Co. N 1511-250) was dissolvedin 2 ml of Bis Tris, pH 6.5 and 8 ml of water.

Luciferol solution: approximately 4 mg of luciferol was dissolved in 1ml of dimethylformamide (Aldrich 27, 054-7).

Luciferase detection solution: A bottle of P450 Glo Buffer (PromegaCorp. V865B) was used to dissolve a cake of Luciferin Detection Reagent(Promega Corp. V859B) then 10 ml of the resulting solution was dilutedwith 8 ml of water to create the detection solution used in thisexample.

The reaction solutions shown in Table 15 were assembled.

TABLE 15 500 mM BisTris, pH NAD+ Y Reaction 6.5 Sol'n Water Eq ADH ADHAl DH Rx 1 20 μl 20 μl 155 μl 0 μl 0 μl 0 μl Rx 2 20 μl 20 μl 145 μl 5μl 0 μl 5 μl Rx 3 20 μl 20 μl 145 μl 0 μl 5 μl 5 μl Rx 4 20 μl 20 μl 150μl 5 μl 0 μl 0 μl Rx 5 20 μl 20 μl 150 μl 0 μl 5 μl 0 μl Rx 6 20 μl 20μl 150 μl 0 μl 0 μl 5 μlA 5 μl sample of the luciferol DMF solution was added to each of thereactions above and a timer was started upon the addition of the firstluciferol/DMF solution to reaction 1. All reactions were kept at roomtemperature.

As each reaction reached 10 minutes post luciferol addition, a 10 μlsample of each reaction was added to 90 μl of luciferin detectionsolution in a luminometer tube. After incubating 5 minutes at roomtemperature, the light produced by these samples was measured using aTurner TD 20 luminometer. Additional samples were taken and mixed withluciferin detection reagent and read as above as each reaction reached20 and 40 minutes post luciferol addition.

TABLE 16 Time Rx 1 Rx 2 Rx 3 Rx 4 Rx 5 Rx 6 10 8.452 9.857 7.481 11.407.396 7.640 minutes 20 10.04 13.91 8.292 15.26 8.670 7.640 minutes 408.406 16.80 8.465 19.16 8.194 8.475 minutesResults

Comparison of light values generated from samples of the reactionwithout enzyme (reaction 1) to that with equine ADH with Al DH reaction2) or alone

(reaction 4) demonstrated that a solution of equine alcoholdehydrogenase and NAD+ can convert luciferol into a form that is moreeffectively used for light production (Table 16). However, samples fromsolutions of yeast alcohol dehydrogenase (with NAD+) alone (reaction 5)or with Al DH (reaction 3) or Al DH alone (reaction 6) showed nosignificant light production above that seen for reactions not givenenzyme (reaction 1).

The results are somewhat surprising as: 1) alcohol dehydrogenase usuallyproduces an aldehyde product and such a product is not thought to be aneffective luciferase substrate, and 2) a second alcohol dehydrogenase,from yeast, was not able to convert sufficient luciferol to beeffectively measured even in the presence of aldehyde dehydrogenase, anenzyme that is capable of transforming aldehydes to the correspondingacids.

B. Conversion of Luciferol by Alcohol Dehydrogenase

To determine whether enzyme concentration or temperature alter thekinetics of a reaction between different ADHs and a luciferinderivative, reactions similar to those in Example 2A were conducted, andlight production over a longer period of time measured.

Materials and Methods

Buffer; KPO₄ Buffer: A 500 mM solution of KPO₄, pH 7.4 was produced bydissolving KH₂PO₄ in water and adjusting the pH with KOH.

NAD+; Solid NAD+, 66 mg (Sigma Chem. Co. N1511-250) was dissolved in 10ml of 50 mM KPO₄, pH 7.4.

The equine ADH, yeast ADH and aldehyde dehydrogenase and luciferolsolutions were those described in Example 5.

Luciferin detection reagent: A bottle of P450 Glo Buffer (Promega Corp.V865B) was used to dissolve a cake of Luciferin Detection Reagent(Promega Corp. V859B) then 5 ml of the resulting solution was dilutedwith 3 ml of water to create the detection solution used in thisexample.

Table 17 summarizes the assembled reactions.

TABLE 17 Reaction NAD+ sol'n KPO₄ Buffer Water Enzyme Rx 1 and 11 20 μl18 μl 157 μl None Rx 2 and 12 20 μl 18 μl 147 μl 10 μl equine ADH Rx 3and 13 20 μl 18 μl 137 μl 20 μl equine ADH Rx 4 and 14 20 μl 18 μl 127μl 30 μl equine ADH Rx 5 and 15 20 μl 18 μl 117 μl 40 μl equine ADH Rx 620 μl 18 μl 112 μl 40 μl equine ADH Rx 7 20 μl 18 μl 152 μl  5 μl yeastADH Rx 8 20 μl 18 μl 147 μl 10 μl yeast ADH Rx 9 20 μl 18 μl 137 μl 20μl yeast ADH Rx 10 20 μl 18 μl 132 μl 20 μl yeast ADH

To initiate the conversions, 5 μl of luciferol solution was added toreactions 1-5, 6-9, and 11-15, and 10 μl of luciferol was added toreactions 5 and 10 and the tubes mixed after that addition. Immediatelyafter luciferol addition to reaction 1, a timer was started and the timeof luciferol addition to the other tubes noted. After mixing, reaction11-15 were placed at 37° C.

As each tube reached 30 minutes post luciferol addition, 5 μl samples ofthe reaction were removed, added to 95 μl of luciferin detection reagentand the tube mixed. After incubating 10 minutes at room temperature, thelight produced by these samples was read using a Turner TD 20luminometer (Table 18). Samples were also taken at 60, 95 190 and 280minutes post luciferol addition and mixed with luciferin detectionreagent and read (Table 18).

TABLE 18 30 190 280 Reaction minutes 60 minutes 95 minutes minutesminutes Rx 1 6.077 6.091 5.849 4.364 7.694 Rx 2 13.86 21.41 29.38 50.8874.52 Rx 3 28.21 49.00 69.94 123.5 166.5 Rx 4 35.79 62.64 94.04 163.9245.9 Rx 5 42.30 80.23 120.5 199.8 297.6 Rx 6 44.49 83.19 123.8 210.2298.1 Rx 7 6.184 6.052 5.980 6.640 7.988 Rx 8 6.126 5.468 5.552 6.1986.903 Rx 9 5.406 5.608 6.301 5.825 6.734 Rx 10 9.672 9.531 10.00 10.3611.71 Rx 11 5.814 6.078 6.302 6.889 8.399 Rx 12 71.30 119.4 151.5 224.0286.5 Rx 13 127.0 213.3 293.1 461.0 619.7 Rx 14 161.7 292.1 433.1 682.2844.5 Rx 15 192.7 365.4 525.7 842.8 1138Results

Solutions of equine alcohol dehydrogenase and NAD+ converted luciferolto a form more effectively used by luciferase for light production. Thestrength of the light signal produced was dependent upon the length oftime used in the conversion reaction (comparing light signals fromreaction 5 at 30, 60, 95, 190 and 280 minutes and the amount of equineADH added and comparing signals of reactions 1-5 over time). Inaddition, a much faster rate of signal rise was seen when the reactionswere incubated at 37° C. versus room temperature (compare pairedreactions such as reaction 5 and reaction 15). Finally, doubling theluciferol concentration in the reactions with equine ADH did notincrease the signal strength greatly (reaction 5 versus 6), and no majorlight signal change was seen with yeast alcohol dehydrogenase. Thus, therate of conversion of luciferol by alcohol dehydrogenase was shown to bedependent upon the amount of the enzyme present in the reaction solutionand the temperature of the reaction.

C. Effect of Inhibitors on the Conversion of Luciferol by Equine AlcoholDehydrogenase

As discussed herein, bioluminogenic reactions with luciferin derivativescan be employed to detect agents that modulate a nonluciferase-mediatedreaction or a luciferase-mediated reaction. A known inhibitor of equineADH was tested in a bioluminogenic reaction having the luciferinderivative luciferol.

Materials and Methods

A solution of equine alcohol dehydrogenase (Sigma Chemical Corp., A9589) 15 mg was dissolved in 750 μl of water.

Pyrazol Solution: 3.4 mg of pyrazol (Sigma Chemical Corp. P 5660-5) wasdissolved in 50 ml of water.

Trichloroethanol solution: A 0.48 ml sample of 2,2,2, trichloroethanol(Aldrich T5480-5) was dissolved in water.

The other solutions used are described in Examples 5-6.

Duplicate reactions were assembled (see Table 19).

TABLE 19 KPO₄ Equine Reaction NAD+ Buffer Water ADH Other Control 10 μl9 μl 59.5 μl 20 μl None +Pyrazol 10 μl 9 μl 49.5 μl 20 μl 10 μl pyrazol+Trichloro- 10 μl 9 μl 49.5 μl 20 μl 10 μl ethanol trichloroethanolResults

Reactions were initiated by addition of 1.5 μl of luciferol solution.Upon addition of the luciferol solution, the reactions were mixed andplaced at 37° C. After 30 minutes, a 5 μl sample was removed from eachreaction, mixed with 95 μl of luciferin detection reagent and the lightproduced by these solutions was measured using a Turner TD 20luminometer after allowing the solutions to sit 10 minutes at roomtemperature. The following light readings were recorded (Table 20).

TABLE 20 Reaction Relative Light Units Control #1 1023 Control #2 1087+Pyrazol #1 516.4 +Pyrazol #2 490.7 +trichloroethanol #1 524.8+trichloroethanol #2 342.1

Thus, the conversion of luciferol by solutions of equine alcoholdehydrogenase and NAD+ was slowed by known inhibitors of equine alcoholdehydrogenase. This reduction in the rate of conversion can be used toidentify agents effecting this enzyme.

Example 3 MAO and FMO Assays with Derivatives of the Invention

A. Luciferin Derivatives for MAO Assays

A series of luciferin derivatives was prepared as substrates formonoamine oxidases (MAO), and those substrates tested in two step assayswith different types of MAO (MAO A and MAO B). Additional assays wereconducted with test compounds to determine whether the compoundinhibited the MAO mediated reaction. Also, as described hereinbelow,fluorogenic MAO substrates were prepared and tested in a one step assay.The bioluminogenic and fluorogenic MAO substrates may be oxidized bymonoamine oxidase (MAO) A and/or B, e.g., to produce iminium and/oraldehyde intermediates that may undergo a secondary β-elimination toliberate 7-hydroxyluciferins or a derivative thereof, or hydroxyfluorescent products including umbelliferone, fluorescein, and theirderivatives. The formation of these products is measured by light outputfrom bioluminescence, where the product of the reaction between MAO anda luciferin derivative is oxidized by luciferase(s), or fromfluorescence. Bioluminogenic and/or fluorogenic monoamine oxidasesubstrates and their relevant compositions provide a highly sensitiveand convenient tool for detecting monoamine oxidase activities. Thus,these derivatives are very useful for high throughput screening formonoamine oxidase substrates and inhibitors thereof.

Materials and Methods

In the reactions described in this example, a luciferin derivative wasincubated with MAO (Step 1). This may generate a species that mayundergo spontaneous beta-elimination to form luciferin.

Alternatively, the product of the reaction between the derivative andMAO yields a product that is not D-luciferin but is a substrate ofluciferase. A reconstituted luciferin detection reagent was then added(Step 2), and the light production from the mixture was measured witheither a Dynex or VERITAS™ plate luminometer.

For instance, to prepare a compound of formula CCLIV, a solution ofdimethylamine (0.235 g, 0.00090 mol) and D-cysteine (0.158 g, 0.00090mol) in methanol (5 ml), CH₂Cl₂ (1 ml) and H₂O (1 ml) was added K₂CO₃(0.125 g, 0.00090 mol). The mixture was stirred at room temperature for5 minutes and then neutralized to a slightly acidic condition. Afterremoval of organic solvent, the product was purified by HPLC using 0.1%TFA water/acetonitrile as eluent. The compound was confirmed by 1HNMR/MS. The yield was 0.23 g. The compound was characterized by 1H NMRand Mass spectra. Related compounds include a compound of formula CCLV:

The reconstituted luciferin detection reagent for Step 2 was generatedby thawing a bottle of P450-Glo Buffer (V865A, Promega Corp., MadisonWis.), allowing it to come to room temperature, and then adding it to abottle of Luciferin Detection Reagent (V859A, Promega Corp., MadisonWis.) with 100 μl of 20% dodecyltrimethylammonium bromide (DTAB)(D-8638, Sigma Chemical Company). The DTAB was added to inactivate MAO.

Results

MAO B and MAO-3

In this example, substrate MAO-3 (FIG. 13; stock=8.19 mM in DMSO) wasserially diluted in DMSO, and 5 μl aliquots of each substrate dilutionwere placed in separate wells of a white, opaque 96-well plate. Toinitiate Step 1, 45 μl of enzyme and buffer were added to each well.This 45 μl contained 10 μl 1 M Tricine pH 8.3, 5 μl 200 mM MgSO₄, 29 μlH₂O, and 1 μl of 5 mg/ml microsomes containing human recombinantmonoamine oxidase B (MAO B) expressed in baculovirus infected insectcells (M-7441, Sigma Chemical Company). Step 1 was incubated at roomtemperature for one hour. Step 2 was initiated by adding 50 μl of thereconstituted luciferin detection reagent to each well. After 20minutes, the luminescent signal from the 100 μl reaction was read on aVERITAS™ plate luminometer. To measure the activity of luciferasetowards the luciferin derivative, the luminescent signal was also readfrom control wells in which MAO B was substituted with H₂O in Step 1.

The relative light units (RLUs) measured are shown in Table 21.

TABLE 21 [MAO-3] (μM) no MAO MAO B net RLUs 819.00 28342 54262 25920409.50 13966 37602 23636 204.75 7294 29701 22407 102.38 3535 24242 2070751.19 1757 20078 18321 25.59 1070 14104 13034 12.80 522 9317 8795 6.40284 5493 5209 3.20 215 2973 2758 1.60 138 1617 1479 0.80 112 962 850A fit of this data in TableCurve Windows v1.0 (Jandel Scientific, AISNSoftware) yielded a K_(m) value of 24±1 μM and a signal-to-backgroundratio at the K_(m) value of about 13.MAO A and MAO-7

In this example, microsomes (5 mg/ml) containing human recombinantmonoamine oxidase A (MAO A) expressed in baculovirus infected insectcells (M-7316, Sigma Chemical Company) were diluted 10-fold in 100 mMTricine pH 8.3, and 10 μl aliquots of the diluted microsomes were placedin separate wells of a white, opaque 96-well plate. Substrate MAO-7(FIG. 13; stock=23.2 mM in DMSO) was serially diluted in DMSO. Toinitiate Step 1, 40 μl of substrate and buffer were added to each well.This 40 μl contained 10 μl 1 M Tricine pH 8.3, 5 μl 200 mM MgSO₄, 15 μlH₂O, and 10 μl of each substrate dilution. Step 1 was incubated at roomtemperature for one hour. Step 2 was initiated by adding 50 μl of thereconstituted luciferin detection reagent to each well. After 20minutes, the luminescent signal from the 100 μl reaction was read on aDynex plate luminometer. To measure the activity of luciferase towardsthe luciferin derivative, the luminescent signal was also read fromcontrol wells in which MAO A was substituted with H₂O in Step 1.

The relative light units (RLUs) measured are shown in Table 22.

TABLE 22 [MAO-7] (μM) no MAO MAO A net RLUs 4640 11.504 650.305 638.8012320 6.0332 542.257 536.2238 1160 3.2977 390.469 387.1713 580 1.6627224.458 222.7953 290 0.8456 124.435 123.5894 145 0.4563 64.975 64.518772.5 0.2556 32.52 32.2644 36.3 0.1312 16.821 16.6898 18.1 0.1033 8.53388.4305A fit of this data in TableCurve Windows v1.0 (Jandel Scientific, AISNSoftware) yielded a K_(m) value of 1560±130 μM and asignal-to-background ratio at the K_(m) value of about 95.MAO A and MAO-3, MAO A and MAO-7, MAO B and MAO-3, MAO B and MAO-7

In this example, microsomes (5 mg/ml) containing human recombinantmonoamine oxidase A (MAO A) expressed in baculovirus infected insectcells (M-7316, Sigma Chemical Company) were diluted 10-fold in 200 mMTricine pH 8.3, and 10 μl aliquots of the diluted microsomes were placedin separate wells of a white, opaque 96-well plate. Substrates MAO-3(stock=9.2 mM in 0.2% H₂SO₄/DMSO) and MAO-7 (stock=26 mM in 0.2%H₂SO₄/DMSO) were serially diluted in 0.2% H₂SO₄/DMSO. To initiate Step1, 40 μl of substrate and buffer were added to each well. This 40 μlcontained 10 μl 1 M Tricine pH 8.3, 5±200 mM MgSO₄, 5 μl 10% TergitolNP-9, 10 μl H₂O, and 10 μl of each substrate dilution. Step 1 wasincubated at room temperature for one hour. Step 2 was initiated byadding 50 μl of the reconstituted luciferin detection reagent to eachwell. After 20 minutes, the luminescent signal from the 100 μl reactionwas read on a Dynex plate luminometer. To measure the activity ofluciferase towards the luciferin derivative, the luminescent signal wasalso read from control wells in which MAO A or MAO B was substitutedwith H₂O in Step 1.

The relative light units (RLUs) measured are shown in Tables 23-24.

TABLE 23 no [MAO-3] (μM) MAO MAO A MAO B net A net B 1840 34.224 155.693362.398 121.469 328.174 920 19.128 108.18 231.563 89.052 212.435 46010.157 65.297 143.784 55.14 133.627 230 4.8948 33.833 77.778 28.938272.8832 115 2.4175 17.977 40.089 15.5595 37.6715 57.5 1.2856 9.477720.741 8.1921 19.4554 28.8 0.5877 4.5843 10.46 3.9966 9.8723 14.4 0.33382.1812 5.5996 1.8474 5.2658 7.2 0.1692 1.2114 2.7601 1.0422 2.5909 3.60.1074 0.6851 1.6472 0.5777 1.5398

TABLE 24 [MAO-7] (μM) no MAO MAO A MAO B net A net B 5200 10.65 553.5152.972 542.86 42.322 2600 6.7022 490.271 43.713 483.5688 37.0108 13003.8297 342.71 39.242 338.8803 35.4123 650 2.1547 196.223 26.367 194.068324.2123 325 1.189 97.309 16.235 96.12 15.046 162.5 0.5589 50.033 9.146349.4741 8.5874 81.3 0.3343 22.845 5.0852 22.5107 4.7509 40.6 0.164111.762 2.7455 11.5979 2.5814 20.3 0.0942 6.1583 1.4467 6.0641 1.352510.2 0.0754 2.8488 0.7627 2.7734 0.6873

A fit of this data in TableCurve Windows v1.0 (Jandel Scientific, AISNSoftware) yielded the RLU_(max) and K_(m) values shown in Table 25. Asindicated by the ratio RLU_(max)/K_(m), MAO-3 is a more specificsubstrate for MAO B and MAO-7 is a more specific substrate for MAO A.

TABLE 25 RLU_(max) K_(m) (mM) RLU_(max)/K_(m) (mM⁻¹) MAO A & MAO-3 2101.32 159 MAO B & MAO-3 660 1.88 351 MAO A & MAO-7 750 1.7 441 MAO B &MAO-7 48 0.65 74MAO A and MAO-11, and MAO B and MAO-11

In this example, MAO-11 (FIG. 13), the methyl ester derivative of MAO-3,was synthesized in an effort to increase enzyme specificity bydecreasing the charge of the substrate and lowering the K_(m) values ofthe MAO enzymes. Microsomes (5 mg/ml) containing human recombinantmonoamine oxidase A (MAO A) or B (MAO B) expressed in baculovirusinfected insect cells (M-7316 and M-7441, respectively, Sigma ChemicalCompany) were diluted 5-fold in 100 mM HEPES pH 7.4, and 5 μl aliquotsof the diluted microsomes were placed in separate wells of a white,opaque 96-well plate. Substrate MAO-11 (stock=16.4 mM in DMSO) wasserially diluted in DMSO. To initiate Step 1, 45 μl of substrate andbuffer was added to each well. This 45 μl contained 10 μl 500 mM HEPESpH 7.4, 5 μl 200 mM MgSO₄, 28 μl H₂O, and 2 μl of each substratedilution. Step 1 was incubated at room temperature for one hour. Step 2was initiated by adding 50 μl of the reconstituted luciferin detectionreagent to each well; 0.5 μl of 3.57 units/μl porcine liver esterase(E-2884, Sigma Chemical Company) was also added to each well to removethe ester prior to reaction with luciferase. After 30 minutes, theluminescent signal from the 100 μl reaction was read on a VERITAS™ plateluminometer. To measure the activity of luciferase towards the luciferinderivative, the luminescent signal was also read from control wells inwhich MAO A or MAO B was substituted with H₂O in Step 1.

The relative light units (RLUs) measured are shown in Table 26.

TABLE 26 [MAO-11] (μM) no MAO MAO A MAO B net A net B 656 117465 8633713551608 8516248 434143 328 62791 8924217 515953 8861426 453162 164 330568079844 509311 8046788 476255 82 17316 6575618 466321 6558302 449005 4110457 4799614 467175 4789157 456718 20.5 6653 2856723 454410 2850070447757 10.25 3671 1136043 348002 1132372 344331 5.13 2084 475853 251284473769 249200 2.56 1271 211752 183244 210481 181973 1.28 823 94496114581 93673 113758 0.64 647 44547 67136 43900 66489 0.32 441 1997331996 19532 31555

A fit of this data in TableCurve Windows v1.0 (Jandel Scientific, AISNSoftware) yielded K_(m) values of 47±6 μM and 4.4±0.4 μM andsignal-to-background ratios at the K_(m) values of about 450 and about130 for MAO A and MAO B, respectively. This represents 33-fold and5.5-fold decreases in the K_(m) values for MAO A and MAO B,respectively, and demonstrates the advantage of using esterifiedsubstrates in Step 1 and including esterase in Step 2.

Measurement of IC₅₀ Values

In this example, test compounds were serially diluted in H₂O, and 12.5μl aliquots of each dilution were placed in separate wells of a white,opaque 96-well plate. Substrate MAO-11 was diluted (stock=16.4 mM) in200 mM HEPES, 10% glycerol, pH 7.4 (+20% dimethyl sulfoxide for MAO B),and 12.5 μl aliquots of 160 μM or 36 μM MAO-11 were added to each wellfor reactions with MAO A or MAO B, respectively. Microsomes (5 mg/ml)containing human recombinant monoamine oxidase A (MAO A) or B (MAO B)expressed in baculovirus infected insect cells (M-7316 and M-7441,respectively, Sigma Chemical Company) were diluted 25-fold in 100 mMHEPES, 5% glycerol, pH 7.4 (+10% dimethyl sulfoxide for MAO B), and toinitiate Step 1, 25 μl aliquots of the diluted microsomes were added toeach well. Step 1 was incubated at room temperature for one hour. Step 2was initiated by adding 50 μl of a modified, reconstituted luciferindetection reagent to each well. In this modified reagent, a bottle ofLuciferin Detection Reagent (V859A, Promega Corp., Madison Wis.) wasdissolved in 200 mM PIPES, pH 6.7, 6.8 mM

MgSO₄, 2% Tergitol NP-9, 0.2% dodecyltrimethylammonium bromide (DTAB)(D-8638, Sigma Chemical Company), 0.2% Mazu DF-204, 18 μM2-(4-aminophenyl)-6-methylbenzthiazole (APMBT), and 20 U/ml porcineliver esterase (E-2884, Sigma Chemical Company). After 30 minutes, theluminescent signal from the 100 μl reaction was read on a Dynex plateluminometer. To measure the activity of luciferase towards the luciferinderivative, the luminescent signal was also read from control wells inwhich MAO A or MAO B was substituted with 100 mM HEPES, 5% glycerol, pH7.4 (+10% dimethyl sulfoxide for MAO B) in Step 1.

The relative light units (RLUs) measured are shown in Tables 27-29.

TABLE 27 MAO A MAO B percent activity [CLOR] no no MAO (mM) enzymeenzyme enzyme enzyme A MAO B 250.0000 20.839 25.21 96.566 4.2724 −0.00911.286 83.3333 20.754 19.884 231.028 4.0911 −0.010 27.679 27.7778 22.49619.544 460.408 3.992 0.018 55.645 9.2593 27.131 20.336 748.578 4.0160.091 90.778 3.0864 35.455 20.883 799.833 3.8485 0.224 97.027 1.028866.13 21.173 824.099 4.052 0.714 99.985 0.3429 125.226 21.205 835.2014.2169 1.657 101.339 0.1143 359.688 20.964 848.71 3.8082 5.399 102.9860.0381 4403.78 20.538 855.032 3.8881 69.939 103.756 0.0127 5556.0720.811 827.772 3.8107 88.329 100.433 0.0042 5848.05 21.288 828.4243.9595 92.988 100.512 0.0014 6287.39 24.955 824.222 4.028 100.000100.000

TABLE 28 MAO A MAO B percent activity [DEP] no no MAO (mM) enzyme enzymeenzyme enzyme A MAO B 125.00000 389.273 25.21 7.0808 4.2724 5.667 0.37941.66667 1421.31 19.884 12.698 4.0911 21.565 1.071 13.88889 2941.0919.544 25.154 3.992 44.977 2.603 4.62963 4928.34 20.336 55.661 4.01675.590 6.357 1.54321 6019.12 20.883 129.138 3.8485 92.393 15.399 0.514406375.81 21.173 290.883 4.052 97.888 35.303 0.17147 6702.08 21.205521.983 4.2169 102.914 63.742 0.05716 6549.63 20.964 704.981 3.8082100.565 86.261 0.01905 6607.84 20.538 789.888 3.8881 101.462 96.7090.00635 6810.54 20.811 825.09 3.8107 104.584 101.041 0.00212 6494.6521.288 805.968 3.9595 99.718 98.688 0.00071 6512.94 24.955 816.628 4.028100.000 100.000

TABLE 29 MAO A MAO B percent activity [PEA] no no MAO (mM) enzyme enzymeenzyme enzyme A MAO B 13650.000 84.655 25.21 7.5654 4.2724 0.986 0.4174550.000 282.629 19.884 11.202 4.0911 4.072 0.842 1516.667 789.16719.544 24.813 3.992 11.969 2.434 505.556 2001.33 20.336 57.877 4.01630.866 6.301 168.519 3890.4 20.883 130.845 3.8485 60.315 14.833 56.1735520.26 21.173 322.368 4.052 85.723 37.230 18.724 6127.62 21.205 623.724.2169 95.192 72.470 6.241 6374.2 20.964 757.033 3.8082 99.036 88.0602.080 6623.63 20.538 827.386 3.8881 102.924 96.287 0.693 6497.46 20.811822.64 3.8107 100.957 95.732 0.231 6497.21 21.288 855.314 3.9595 100.95399.553 0.077 6436.06 24.955 859.135 4.028 100.000 100.000

A fit of this data in TableCurve Windows v1.0 (Jandel Scientific, AISNSoftware) yielded IC₅₀ values that match published values (see Table30).

TABLE 30 published values IC₅₀ (μM) (μM) compound MAO A MAO B MAO A MAOB clorgyline (CLOR) 0.024 ± 0.007 24 ± 5  0.025 79 deprenyl (DEP) 7.1 ±0.8 0.151 ± 0.008 5 0.13 phenethylamine 134 ± 9  20 ± 2  78-280 2-150(PEA)B. Assays with a Fluorogenic Derivative

In the following example, reactions were assembled in which a substrateexhibiting low fluorescence was incubated with MAO. This may generate aspecies that may undergo spontaneous β-elimination to form a productexhibiting high fluorescence. The fluorescent signal was then measured(excitation/emission=355/460 nm) on a FLUOROSKAN ASCENT™ platefluorometer.

Microsomes containing human recombinant monoamine oxidase A (MAO A) or B(MAO B) expressed in baculovirus infected insect cells (M-7316 andM-7441, respectively, Sigma Chemical Company) were diluted 10-fold in200 mM Tricine pH 8.3, and 10 μl aliquots of the diluted microsomes wereplaced in separate wells of a black 96-well plate. Substrate MAO-F3(FIG. 11; stock=69.1 mM in DMSO) was serially diluted in DMSO. Toinitiate the reaction, 90 μl of substrate and buffer was added to eachwell. This 90 μl contained 35 μl mM Tricine pH 8.3, 35 μl 200 mM TricinepH 8.7 (or water), 10 ml 200 mM MgSO₄, and 10 μl of each substratedilution. The 100 μl reaction was incubated at room temperature for onehour and the fluorescent signal was read on a FLUOROSKAN ASCENT™ platefluorometer. To measure the fluorescence of the derivative, thefluorescent signal was also read from control wells in which MAO A orMAO B was substituted with H₂O.

The relative fluorescent signals measured are shown in Table 31.

TABLE 31 [MAO-F3] (μM) no MAO MAO A MAO B net A net B 863.75 6761 2115417322 14393 10561 431.88 5097 19530 16590 14433 11493 215.94 3375 1672114149 13346 10774 107.97 2037 13930 11704 11893 9667 53.98 1210 115559885 10345 8675 26.99 633 8461 7468 7828 6835 13.50 348 6078 5239 57304891 6.75 197 4129 3683 3932 3486 3.37 118 2604 2198 2486 2080 1.69 781486 1353 1408 1275 0.84 57 815 689 758 632 0.42 48 431 375 383 327 0.2148 240 202 192 154

A fit of this data in TableCurve Windows v1.0 (Jandel Scientific, AISNSoftware) yielded K_(m) values of 21±1 μM and 16±1 μM andsignal-to-background ratios at the K_(m) values of about 15 and about 13for MAO A and MAO B, respectively.

Conclusion

Thus, luciferin and fluorophore derivatives allow MAO (see FIGS. 11 and13) to be measured through conversion of the derivative. Conversion ofthe derivative may not directly generate native luciferin or afluorogenic molecule, but may yield an intermediate that converts toluciferin or the fluorogenic molecule, or produces an intermediate thatis a substrate for luciferase. Such compounds are useful to measure MAO,or inhibitors or substrates for MAO. One luciferin derivative tested(fluoroluciferin) gave a non-pH dependent light signal.

In particular, luciferin derivatives such as MAO #1, 3 and the like weretransformed by monoamine oxidases A and B to a product that may beutilized by luciferases much more effectively than the derivative,allowing these enzymes to be measured easily and effectively. Some ofthese compounds, such as MAO #1, #3, #4 (FIG. 13), were designed suchthat the monoamine oxidase reaction would generate a product that wouldbe able to spontaneously cleave off the luciferin nucleus in anelimination reaction. The data suggested that this elimination reaction,if it takes place, surprisingly takes place very rapidly. Such areaction thus allows for the measurement of the MAO enzymes without theneed for a second incubation step for the elimination reaction to go tocompletion. Additional derivatives were generated that were designed toincrease the rate of the elimination reaction by inclusion of a fluorideatom on the luciferin nucleus. Such an enhancement may be generallyuseful in other elimination reactions. Moreover, the product of thosereactions may be utilized as a luciferase substrate.

C. Luciferin Derivatives for FMO Assay

In the following examples, reactions were assembled in which a luciferinderivative was incubated with FMO (Step 1). This may generate a speciesthat may undergo spontaneous beta-elimination to form luciferin. Areconstituted luciferin detection reagent (see previous examples) wasthen added (Step 2), and the light production from the mixture wasmeasured with either a Dynex or VERITAS™ plate luminometer.

FMO 1 and FMO 3 and substrate FMO-2

In this example, 1 μl aliquots of microsomes containing humanrecombinant flavin-containing monooxygenase 1 (FMO 1) or 3 (FMO 3)expressed in baculovirus infected insect cells (F-4928 and F-5053,respectively, Sigma Chemical Company) were placed in separate wells of awhite, opaque 96-well plate. To initiate Step 1, 49 μl of substrate andbuffer was added to each well. This 49 μl contained 5 μl 200 mM CHES pH9.5, 2.5 μl Solution A, 0.5 μl Solution B, 39 μl H₂O, and 2 μl of 3.75mM substrate FMO-2. Solution A and Solution B are components of theNADPH Regeneration System (V9510, Promega Corp., Madison Wis.). SolutionA contains 26 mM NADP⁺, 66 mM glucose-6-phosphate, and 66 mM MgCl₂.Solution B contains 40 units/ml glucose-6-phosphate dehydrogenase in 5mM citrate pH 5.5. Step 1 was incubated at room temperature for fourhours. Step 2 was initiated by adding 50 μl of the reconstitutedluciferin detection reagent to each well. After 30 minutes, theluminescent signal from the 100 μl reaction was read on a Dynex plateluminometer. To measure the activity of luciferase towards the luciferinderivative, the luminescent signal was also read from control wells inwhich FMO 1 or FMO 3 was substituted with microsomes from wild typebaculovirus infected insect cells (M-7566, Sigma Chemical Company) inStep 1.

The relative light units (RLUs) measured are shown in Table 32.

TABLE 32 sample control FMO 1 FMO 3 RLUs 8.7102 8.5489 11.778 12.00612.194 10.425FMO 3 and Substrate MAO-7

In this example, 5 μl aliquots of microsomes containing humanrecombinant flavin-containing monooxygenase 3 (FMO 3) expressed inbaculovirus infected insect cells (F-5053, Sigma Chemical Company) wereplaced in separate wells of a white, opaque 96-well plate. SubstrateMAO-7 (FIG. 13, stock=17.2 mM in DMSO) was serially diluted in DMSO.

To initiate Step 1, 45 μl of substrate and buffer was added to eachwell. This 45 μl contained 10 μl 1200 mM CHES pH 9.5, 2.5 μl Solution A,0.5 μl Solution B, 30 μl H₂O, and 2 μl of each substrate dilution.Solution A and Solution B are components of the NADPH RegenerationSystem (V9510, Promega Corp., Madison Wis.). Solution A contains 26 mMNADP⁺, 66 mM glucose-6-phosphate, and 66 mM MgCl₂. Solution B contains40 units/ml glucose-6-phosphate dehydrogenase in 5 mM citrate pH 5.5.Step 1 was incubated at room temperature for one hour. Step 2 wasinitiated by adding 50 μl of the reconstituted luciferin detectionreagent to each well.

After 30 minutes, the luminescent signal from the 100 μl reaction wasread on a VERITAS™ plate luminometer. To measure the activity ofluciferase towards the luciferin derivative, the luminescent signal wasalso read from control wells in which FMO 3 was substituted withmicrosomes from wild type baculovirus infected insect cells (M-7566,Sigma Chemical Company) in Step 1. The relative light units (RLUs)measured are shown in Table 33.

TABLE 33 [MAO-7] (μM) control FMO 3 net FMO 3 688 7213 42707 35494 5166137 40329 34192 344 4486 34036 29550 258 3592 28987 25395 172 257122463 19892 129 2004 19332 17328 86 1585 13646 12061 43 1043 8220 7177A fit of this data in TableCurve Windows v1.0 (Jandel Scientific, AISNSoftware) yielded a K_(m) value of 200±20 μM and a signal-to-backgroundratio at the K_(m) value of about 8.5. FIGS. 14-15 provide exemplary FMOsubstrates.

Example 4 Luciferin Derivatives as Substrates of Alkaline Phosphatase

In this example, AP4, a luciferin derivative, was reacted with variousamounts of calf intestinal alkaline phosphatase in the presence ofluciferase and ATP and light production recorded as a way of measuringalkaline phosphatase activity.

Materials and Methods

Luciferin derivative AP 4, was dissolved in 50 mM Tris Cl pH 7.5 toproduce a 4.3 mM solution of the compound.

Alkaline Phosphatase (Promega Corp. M1821) was diluted in 50 mM Tris Cl,pH 9.3, 1 mM MgCl₂, 0.1 mM ZnCl₂ to a concentration of 1 fmol/μl to 0.01zeptomole/μl.

A reaction solution was assembled containing 1 μM AP 4 (FIG. 69); 100μg/ml of the thermostable luciferase, 2 mM ATP; 10 mM MgCl₂; 100 μMZnCl₂ in 50 mM Tris Cl, pH 8.5. This solution was allowed to incubatefor 1 hour at room temperature to reduce background light production.

Quadruplicate wells in a luminometer plate (Nunc Maxisorb plate) weregiven 10 μl of the diluted alkaline phosphatase stocks and 100 μl ofreaction solution. The plate was incubated at room temperature for 30minutes, then the light produced by the solutions was measured using aVERITAS™ Luminometer.

Results

The following average light readings were calculated from the raw data(Table 34).

TABLE 34 # AP Net molecules Reaction 1 Reaction 2 Reaction 3 Reaction 4Avg Reading Reading 0 2620 2735 2612 2531 2624.5 0 6 2706 2807 2631[lost] 2714.67 89 60 2920 2795 2728 2647 2772.5 148 600 3118 3051 33452805 3079.8 455 6,000 3189 3144 3002 3405 3185 560 60,000 5956 5641 54165728 5685.3 3060 600,000 30042 31132 29781 31679 30658.5 28034 6,000,000281115 254693 265592 296642 274510.5 271886 60,000,000 2639163 22838822320675 2334267 2394497 2391872

Since the signal seen increased incrementally with increasing numbers ofalkaline phosphate molecules, the signal was due to the action ofalkaline phosphate. Exemplary AP substrates are shown in FIG. 16.

Example 5 A. Screening a Human Cytochrome P450 with LuciferinDerivatives

A number of luciferin derivatives useful to detect P450 enzymes in a twostep format were prepared: A, B and C ring modifications on(4S)-4,5-dihydro-2-(6-hydroxybenzothiazolyl)-4-thiazolecarboxylic acid(D-luciferin), and A ring modifications on(4S)-4,5-dihydro-2-(6-aminobenzothiazolyl)-4-thiazolecarboxylic acid(aminoluciferin). In particular, these derivatives were prepared in aneffort to identify luminogenic substrates for P450 enzymes that were notsubstantially active with previously prepared luminogenic substrates(U.S. published application 20040171099) or that showed strongeractivity with certain enzymes. In addition, it was also of interest toidentify substrates with improved selectivity for single P450 enzymes.As described below, in addition to new P450 substrates, luciferin esterswere identified that are luminogenic substrates for carboxylesterases.Luciferin derivatives were tested as substrates for each of a panel ofcytochrome P450 enzymes. Derivatives useful as P450 substrates may beemployed in assays to detect inhibitors for particular P450 enzymes (seeFIGS. 17-18).

A two-step luminescent approach was used to measure P450 activity (Cali,Cell Notes. 7:2 (2003); Cali et al., Cell Notes, 13:8 (2005a); and Cali,Bioluminescent P450 assays that use D-luciferin derivatives assubstrates for CYP1A1, 1A2, 1B1, 2C8, 2C9, 2J2, 3A4, 3A7, 4A11, 4F3B,4F12 and 19, Proc. 14^(th) Int. Conf. Cytochromes P450, Medimond Int.Proc. (2005b)). Briefly, a P450 enzyme reacts with a luciferinderivative to leave D-luciferin as a reaction product. The D-luciferinis then detected in a luciferase reaction mixture, where luminescence inthe reaction is directly proportional to the amount of D-luciferingenerated. A sample that contains active P450 enzyme is compared to acontrol that is devoid of P450 activity. A P450-containing sample thatgives a luminescent signal in significant excess over the control isscored as active without suggesting a reaction mechanism.

Materials and Methods

P450s are Supersomes™ purchased from Discovery Labware (BD/Gentest).These are membrane fractions from an insect cell expression system wherethe P450 has been co-expressed with P450 reductase. CYP2A6, 2B6, 2C8,2C9, 2C19, 2E1, 2J2, 3A4, 3A7, 4F2, 4F3A, 4F3B and 4F12 also containcytochrome b5. HLM is human liver microsomes.

In samples labeled “control”, P450 Supersomes™ or HLM was replaced witha membrane preparation without P450 expression from the insect cellexpression system used for the Supersomes™.

A 2× concentrated P450 reaction mixture for each P450 was prepared withan appropriate buffer and substrate:

-   -   1× buffers:        -   100 mM KPO₄, pH7.4: CYP1A1, 1A2, 1B1, 2D6, 2E1, 3A5, 3A7,            2J2, 4F12, 19, HLM and insect cell control.        -   50 mM KPO₄, pH 7.4: CYP2B6, 2C8, 2C19, 4F2, 4F3A, 4F3B.        -   25 mM KPO₄, pH 7.4: CYP2C9        -   200 mM KPO₄, pH 7.4: CYP3A4 (the KPO₄ is withheld from the            CYP3A4 mix but added with the NADPH regeneration solution in            a subsequent step).        -   100 mM Tris HCl, pH 7.5: CYP2A6, 2C18, 4A11.    -   1× substrate concentration: 50 μM.    -   Each Supersome™ reaction mixture contained 1 pmole P450/50 μL        reaction (except CYP 19 that was used at 5 pmoles/50 μL reaction        where indicated). The HLM reactions contained 20 mg HLM/50 μL        reaction, except where indicated otherwise. Control insect cell        membranes were at 50 μg/50 μL.

A 2×NADPH regeneration solution was prepared as follows for allreactions except CYP3A4: 2.6 mM NADP+, 6.6 mM glucose-6-phosphate, 6.6mM MgCl₂ and 0.8 U/ml glucose-6-phosphate dehydrogenase. A 2×NADPHregeneration solution was prepared for use with CYP3A4: 2.6 mM NADP+,6.6 mM glucose-6-phosphate, 6.6 mM MgCl₂, 0.8 U/ml glucose-6-phosphatedehydrogenase and 400 mM KPO₄.

25 μl of each 2X P450 reaction mixture was added to 3 wells of a whiteopaque 96 well plate. Reactions were initiated by adding 25 μl of the 2XNADPH regeneration solution. The plate was placed in a 37° C. H₂O bathfor 30 minutes. The reactions were stopped and luminescence initiated byadding 50 μl of a P450-Glo™ luciferin detection reagent (LDR) (PromegaCorp.). 20 units/ml of porcine or rabbit esterase (PE) was added to LDRin cases where the P450 luminogenic substrate was a carboxyl ester (seeindividual experiments for an indication of when esterase was included).The plate was moved to room temperature and after 20 minutes,luminescence was read as relative light units (RLU) on a plate readingluminometer (Polarstar Optima by BMG Labtech or VERITAS™ by TurnerBiosystems).

Results

FIGS. 17-18 show data for a panel of recombinant human P450s, HLMs and acontrol insect cell membrane preparation devoid of P450 activity with 41different luciferin derivatives. P450s that converted a derivative to aluminogenic product are identified as those that gave signals that weregreater than the control samples. P450 activity against a derivative isalso indicated by signals from HLM reactions that were greater thancontrol. Lines 11-14 of FIGS. 17-18 show data for 4 different luciferinderivatives with two A ring modifications. The derivative in line 11 ismodified at positions 4 and 6 and is a preferred substrate for isozymes1A1, 1A2, 2C8 and 2C9. The derivative shown in line 12 is also modifiedat positions 4 and 6, and is a preferred substrate for isozymes 1A1,1A2, 2C8 and 2C9. The derivative in line 13 is modified at positions 5and 6, and is a preferred substrate for isozymes 1A2 and 4A11, and to alesser degree, a substrate of 2C8 and 2C9. The derivative in line 14 ismodified at positions 5 and 6, and is a preferred substrate for isozymes2C9 and 4A11

Lines 1-10 of FIGS. 17-18 give RLU for a panel of cytochrome P450enzymes with 10 different single A ring modified luciferin derivatives.All ten derivatives were substrates for P450s, but had different enzymeselectivity profiles. The compounds in lines 7 and 8 are bisluciferins.

Lines 15-20 of FIGS. 17-18 give RLU for a panel of P450 enzymes withluciferin derivatives with a A ring modification and a B ringmodification. Quinolylluciferin 6-methyl ether is a preferred substratefor 1A2 and 4A11, while quinolylluciferin 6-benzyl ether was a preferredsubstrate for 3A7, and to a lesser degree a substrate for 3A4 and 2C8.Naphthylluciferin 6-methyl ether and quinoxalylluciferin 6-methyl etherare a substrate for 4A11.

Lines 21-23 of FIGS. 17-18 show data for aminoluciferin derivatives. Twoof the derivatives were a preferred substrate for 1A1, while the otherderivative was a substrate for 3A7, and to a lesser degree a substratefor 2C8 and 2C9, although background RLUs for that derivative were high.

Lines 24-36 of FIGS. 17-18 show data for derivatives with a C ring and aA ring modification. Derivatives with C and A ring modifications showedsubstantial signals above controls with certain P450 enzymes (e.g.CYP1A1, CYP1A2, CYP2C19, CYP2D6). It was also demonstrated with some ofthese derivatives (luciferin H ethyleneglycol ester, 6-m-picolinylluciferin methyl ester, 6-p-picolinyl luciferin methyl ester, andluciferin 6-methylether propanol ester) that the addition of esterase(+PE) causes substantially more light to be produced than without PE.This indicates that P450s react at the site of the A-ring modificationand that the carboxyl esters (C-ring modifications) are cleaved bycarboxyl esterase activity (PE). With the addition of a C ringmodification to an A ring derivative it was possible to alter the P450enzyme selectivity profile. For example, the compounds in lines 33-36,all C-ring derivatives of luciferin 6 methyl ether (an A ringderivative) showed substantial activity with CYP2D6, whereas luciferin6-methyl ether has an extremely low activity with CYP2D6.

The addition of a picolinyl to the A or C ring of D-luciferin yieldedsubstrates with different specificities for P450 isozymes and RLUs(lines 27 and 28-32 in FIGS. 17-18).

The RLU for some reactions where RLU is less than robust may be enhancedby the addition of esterase (lines 24-25, 28-29, 30-31, 34-35, and 38-39in FIGS. 17-18).

Data for other derivatives and P450 enzymes are also shown in FIGS.20-26, 28-37A, and 38-50.

Showing activity for P450s for which no adequate luminogenic substratecurrently exists were 5,6-dimethoxyluciferin,6(p-aminophenyloxy)-quinolylluciferin and luciferin-6-isobutylcarbonate(lines 14, 17 and 9 in FIGS. 17-18). CYP2A6, CYP2E1 and CYP2C18 areenzymes that reacted with these compounds but not with previouslyavailable luminogenic compounds. 5,6-dimethoxyluciferin and6(p-aminophenyloxy)-quinolylluciferin did not produce large luminescentsignals with any P450, indicating that the enzymes that react with thesesubstrates do so at a slow rate. Luciferin 6-isobutylcarbonate wasnon-selective in that it gave large luminescent signals with severalP450s. This compound also gave a large background signal (minus P450control samples) suggesting that it is chemically unstable in a way thatgives rise to free D-luciferin.

Some compounds showed good selectivity for a single P450 enzyme overother P450 enzymes. Bis-luciferin-methylenediether andbis-luciferin-xylyldiether were selective for CYP1A1 or CYP3A7,respectively. The selectivity of the latter compound for CYP3A7indicates that it would be a highly selective probe substrate forCYP3A7. 6′-p-chlorophenylthiol-methoxy-luciferin (line 2 in FIGS. 17-18)also showed selectivity for CYP3A7 (FIG. 47). CYP3A7 selectivesubstrates may be particularly useful in fetal and neonatal liversamples where CYP3A7 is the dominant P450 and in pediatric samples whereCYP3A7 is gradually replaced over time by CYP3A4 (Stevens et al., J.Pharm. Exp. Ther., 307:573 (2003)). With these substrates CYP3A7activity could be selectively measured against a background of CYP3A4and other P450 activities. The selectivity ofbis-luciferin-methylenediether for CYP1A1 would be particularly usefulfor differentiating CYP1A1 activity from other P450s, especially CYP1A2and CYP1B1, which react with many of the same substrates as CYP1A1 andare also co-expressed with CYP1A1 in certain tissues (Shimada et al.,Drug Metab. Dispos., 29:617 (1997)). Additional compounds weresynthesized that showed improved selectivity for a single P450. Notably,N-benzyloxycarbonyl aminoluciferin and N-isobutoxycarbonyl aminolucifernshowed strong selectivity for CYP1A1 (lines 22-23 in FIGS. 17-18).

The non-carboxylic acid luciferins, luciferin-6-methylether hydrazideand luciferin-6-methylether-methoxylamide showed good selectivity forCYP1A2 over the other P450s tested (lines 40 and 41 in FIGS. 17-18).6′(2,5-ditrifluoromethylbenzyloxy)-luciferin,6′(o-trifluoromethylbenzyloxy)-luciferin,6′(2,3,4,5,6pentafluoro-benzyloxy)-luciferin,6′-(2,3,4,6-tetrafluoro-5-((4-phenylpiperizin-1-yl)methyl)benzyloxy)-luciferin,6′-(3-((4-phenyl-piperizin-1-yl)methyl)benzyloxy)-luciferin,6′(2,4,6-trimethylbenzyloxy)-luciferin and6-benzyloxymethoxy-quinolylluciferin were selective for three members ofthe CYP3A subfamily, CYP3A4, CYP3A5 and CYP3A7 (FIGS. 45-46). This wasan improvement in selectivity for CYP3A over benzyloxy luciferin, whichwas previously shown to react also with CYP4F12 (Cali, 2005b)

The human CYP3A4 enzyme is the most prominent drug-metabolizing P450. Todetermine whether certain luminogenic substrates for CYP3A4 had improvedproperties over the previously identified 3A4 substrates, luciferin6-benzyl ether and luciferin 6′ 3-picolinyl ether, some luciferinderivatives were synthesized and tested for improved selectivity,reduced background luminescence and increased turnover rate, which leadsto brighter signals. The benzyl and picolinyl ethers cross reactedsignificantly with human 3A7 and the benzyl ether also cross reactedwith human CYP4F12. The benzyl ether also showed a high backgroundluminescence that could be from an inefficient light generating reactionwith luciferase that occurs even in the absence of debenzylation byP450.

To improve substrates to assay for CYP3A4, e.g., substrates withincreased stability, reduced background and/or useful in cell-basedassays, luciferin derivatives, some with halogens were prepared (lines 5and 6 in FIGS. 17-18). For instance, modifications to benzyl onluciferin-6-benzylether, e.g., including adding halogen, alkyl, OH orNH₂ to one or more ring atoms, may lead to lower backgroundluminescence. Thus, the following compounds may have increasedspecificity and/or reduced background and may be useful in cell-basedassays.

X═O or NHR1-R5 are independently H, F or

R6 is H, lower alkyl, hydroxyalkyl, or short PEGFor instance, the following were synthesized:

Results

6′(3-((4-phenylpiperizin-1-yl))methyl)benzyloxy) luciferin(6-phenylpiperazinexylyl-luciferin, line 3 in FIGS. 17-18) did not crossreact with 4F12, and its reaction with 3A4 was insensitive to DMSO atcommonly used concentrations. The background luminescence for 6-phenylpiperazinexylyl-luciferin was lower than luciferin-6-benzylether (about2200 versus about 5000 RLU on a BMG luminometer), and signals for6-phenyl piperazinexylyl-luciferin were similar toluciferin-6-benzylether. Moreover, 6-phenyl piperazinexylyl-luciferinwas competitive at 2 of the 3 substrate binding sites on CYP3A4 comparedto luciferin-6-benzylether, which was only competitive at 1 site.Initial results indicated that 6-phenyl piperazinexylyl-luciferin can beemployed in cell-based assays.

6(2,3,4,5,6-pentafluorobenzyl)-luciferin had lower backgroundluminescence than luciferin-6-benzylether and 6-phenylpiperazinexylyl-luciferin (about 900 RLU on a BMG luminometer). Thefluorines may stabilize the compound so that there is less non-enzymaticdegradation or reduce the preference of luciferase for the compoundcompared to luciferin-6-benzylether. Thus, 6-phenylpiperazinexylyl-luciferin may be further stabilized by adding fluorinesto the proximal benzyl group, resulting in a compound with even lowerbackground luminescence.

6(2,4,6 trimethylbenzyl)-luciferin, 6-phenylpiperizinexylyl-luciferin,6-O-trifluoromethylbenzyl luciferin, 6(2,3,4,5,6pentafluorbenzyloxy)-luciferin and6-phenylpiperizine-2,3,5,6-quatrafluoroxylyl-luciferin showedsubstantial activity with CYP3A4 and did not show substantial crossreactivity with CYP4F12 (lines 1-3 and 5-6 of FIGS. 17-18). Some ofthese compounds showed reduced background luminescence and strongersignals with human CYP3A4. Lower background luminescence from thecompounds provides for improved CYP3A assay sensitivity.

Luciferin-6-trifluoromethylbenzylether showed enhanced selectivity forCYP3A5 (line 10 in FIGS. 17-18). 4-methyl-6-(O-methyl)-luciferin and4-(O-methyl)-6-(O-methyl)-luciferin (lines 11-12 in FIGS. 17-18) showedenhanced selectivity for 1A2, 2C8 and 2C9 in that CYP4A11 did not reactwith these compounds in contrast to a previously described compound,luciferin-6-methylether and quinolyl luciferin-6-methyl ether (line 15in FIGS. 17-18) showed enhanced selectivity for CYP4A11 comparedluciferin-6-methylether.

B. Screening Inhibitors of CYP3A4, CYP2D6 and CYP2C19 with LuciferaseDerivative

Materials and Methods

CYP3A4, CYP2D6 and 2C19 Supersomes™ were purchased from DiscoveryLabware (BD/Gentest). “Insect cell control” is a membrane preparationwithout P450 expression from the expression system used for theSupersomes™.

A 4× concentrated P450 reaction mixture for each P450 was prepared withan appropriate buffer and substrate.

-   -   4× CYP2D6 reaction mix: 400 mM KPO₄ (pH 7.4), 140 μM        luciferin-6-methylether ethylene glycol ester (line 36 in FIGS.        17-18), 10 pmoles CYP2D6 Supersomes™/ml.    -   4× CYP2C19 reaction mix: 200 mM KPO₄ (pH 7.4), 100 μM        luciferin-H ethylene glycol ester (lines 24 and 25 in FIGS.        17-18), 10 pmoles CYP2C19 Supersomes™/ml.    -   4× CYP3A4 reaction mix: 200 μM        6(2,3,4,5,6-pentafluorbenzyloxy)-luciferin or        6-phenylpiperizinexylyl-luciferin (lines 3 and 5 in FIGS.        17-18), 40 pmoles CYP3A4 Supersomes™/ml.    -   4× control reaction mix for CYP2D6: 400 mM KPO₄ (pH 7.4), 140 μM        luciferin-6-methylether ethylene glycol ester, 100 μg/ml mg        Supersome™ insect cell control membranes.    -   4× control reaction mix for CYP2C19: 200 mM KPO₄ (pH 7.4), 100        μM luciferin-H ethylene glycol ester, 100 μg/ml Supersome™        insect cell control membranes.    -   4× control reaction mix for CYP3A4 reaction mix: 200 μM        6(2,3,4,5,6-pentafluorbenzyloxy)-luciferin or        6-phenylpiperizinexylyl-luciferin (lines 3 and 5 in FIGS.        17-18), 400 μg/ml Supersomes™ insect cell control membranes.        Luciferin-6-methylether ethylene glycol ester and luciferin-H        ethylene glycol ester were diluted from 10 mM stock solutions        dissolved in acetonitrile, the CYP3A4 substrates from 50 mM        stock solutions dissolved in DMSO.

2X NADPH regeneration solutions were prepared as described above. Testinhibitors were prepared at a 4X concentration. They were diluted in H₂Ofrom stock solution as shown in Table 34. A dilution series of each 4Xinhibitor was prepared in H₂O plus an amount of the vehicle from thestock equivalent to what was carried into the most concentrated 4Xsolution. 0 inhibitor is the stock solution vehicle diluted in H₂O. 12.5μl of each dilution of 4X inhibitor was added to 3 wells of a whiteopaque 96 well for the P450 reactions and 3 wells for the controlreactions. 12.5 μl of each 4X P450 reaction mixture or control reactionmixture was added to the appropriate wells with the 4X inhibitors or 0inhibitor vehicle controls. Plates were placed in a 37° C. H₂O bath for10 minutes. Reactions were then initiated by adding 25 μl of the 2XNADPH regeneration solution. Plates were incubated in the 37° C. H₂Obath for 30 minutes. The reactions were stopped and luminescenceinitiated by adding 50 μl of P450-Glo™ luciferin detection reagent(Promega Corporation) with 20 units/ml of porcine esterase added perreaction. Plates were moved to room temperature and after a 20 minutewait luminescence was read as RLU on a plate reading luminometer(Polarstar Optima by BMG Labtech or VERITAS™ by Turner Biosystems).Luminescence of the control reactions were subtracted from P450reactions and curve fits (sigmoidal dose response with variable slope)and IC₅₀ calculations were performed using the program GraphPad PRISM™.Each inhibitor caused a hyperbolic, dose dependent inhibition with acharacteristic IC₅₀ (Tables 35-36). This data provides examples of howtwo luciferin derivatives each having a left ring modification and a Cring modification can be used in reactions with two different P450enzymes to test for P450 inhibition.

TABLE 35 mM 4X (dilution stock into water), CYP inhibitor solutioncarrier mM 1X carrier conc terfedadine 10 DMSO 0.1 0.25% DMSO quinidine20 H₂O 4 bufuralol 20 H₂O 4 verapamil 20 H₂O 4 clotrimazole 10 DMSO 4  10% DMSO nifedipine 50 DMSO 4   2% DMSO nicardipine 150 DMSO 4 0.67%DMSO Troleandomycin 10 Acetonitrile 0.4   1% acetonitrile bupropion 150DMSO 4 0.67% DMSO haloperidol 50 DMSO 0.1 0.05% DMSO quinine 125 H₂O 4debrisoquine 100 H₂O 4 pindolol 100 DMSO 4   1% DMSO disopyramide 100H₂O 4 (s)-(+)- 50 Acetonitrile 3.2  1.6% mephenytoin acetonitrileFluvoxamine 5 H₂O 0.004 isoniazid 50 H₂O 1.6

TABLE 36 CYP3A4 CYP3A4 6(2,3,4,5,6- 6- CYP2D6 pentafluorbenzyloxy)-phenylpiperizinexylyl- CYP2C19 IC₅₀ luciferin luciferin Inhibitor IC₅₀(μM) (μM) IC₅₀(μM) IC₅₀(μM) terfedadine — 3.6 — — quinidine — 0.01 — —bufuralol — 37 — — verapamil — 67 — — clotrimazole — 22.6 — — nifedipine— 131 — 19.8 nicardipine — 8 — — troleandomycin — — 0.14 — bupropion —33 — — haloperidol — 18 — — quinine — 11 — — debrisoquine — 77 — —pindolol — 76 — — disopyramide — 220 — — (s)-(+)- 69 — — — mephenytoinFluvoxamine 0.25 — — — isoniazid 76 — — —

C. Luciferin Derivatives for P450 Assays

A number of luciferin derivatives useful to detect P450 enzymes in a twostep format were prepared: 6′ ether modifications on(4S)-4,5-dihydro-2-(6-hydroxybenzothiazolyl)-4-thiazolecarboxylic acid(D-luciferin), 6′ modifications on(4S)-4,5-dihydro-2-(6-aminobenzothiazolyl)-4-thiazolecarboxylic acid(aminoluciferin), 6′ modifications on quinolyl luciferin and carboxylesters of D-luciferin, and 6′ modified D-luciferins and 6′ modifiedquinolyl luciferins. In particular, these derivatives were prepared inan effort to identify luminogenic substrates for P450 enzymes that werenot substantially active with previously prepared luminogenic substrates(U.S. published application 20040171099) or that showed strongeractivity with certain enzymes. In addition, it was also of interest toidentify substrates with improved selectivity for single P450 enzymes.As described below, in addition to new P450 substrates, luciferin esterswere identified that are luminogenic substrates for carboxylesterases.

Human P450 enzymes CYP2D6 and CYP2C19 oxidize numerous therapeutic drugscurrently in use (Rendic, Drug Metab. Rev. 34: 83-448 (2002)). Drugdevelopment efforts therefore include screens of new chemical entitiesagainst CYP2D6 and CYP2C19 for modulatory effects of the compounds onthe P450 activities. Thus, there is a need for robust assays for theseenzymes.

Materials and Methods

For FIG. 20, 1 or 5 picomoles of recombinant CYP2D6 or CYP2C19 membranefractions from an insect cell expression system (Supersomes™, DiscoveryLabware) were incubated at 37° C. for 60 minutes with 200 μM D-luciferin6′ methyl ether (luciferin-ME) in 50 mM (CYP2C19) or 100 mM (CYP2D6)KPO₄ (pH 7.4) with an NADPH regenerating system (as described above) ina volume of 50 μl. At the end of the one hour incubation, P450 reactionswere stopped and the detection of P450 generated D-luciferin wasinitiated by adding 50 μl of luciferin detection reagent. For FIG. 20,the panel of human P450s was screened as described above. The luciferindetection reagent (Promega Corp.) was supplemented with 1 unit per 50 μlof porcine liver esterase. Luminescence was read as relative light units(RLU) 20 minutes after the addition of the luciferin detection reagentwith esterase.

Results

Many luciferin derivatives with 6′ modifications were tested assubstrates for CYP2D6 and 2C19 and for use in luminogenic P450 assaysbut significant 2D6 or 2C19 activities were not detected with most ofthese compounds. Luciferin 6-methyl ether did show some activity withthese enzymes, however, the activities were modest in that detectionrequired high substrate and enzyme concentrations and a long incubationtime (FIG. 20).

The literature suggested that D-luciferin derivatives with a carboxylicacid group and its negative charge at pH 7.4 would indeed be poor CYP2D6substrates. Models of the CYP2D6 active site include an aspartic acidresidue; its negative charge would be expected to repel negativelycharged luciferin derivatives (Ellis et al., J. Biol Chem., 270:29055(1995)). To eliminate the negative charge on luciferin-6-methyl ether,the carboxylmethyl ester of luciferin-ME was prepared. P450s might firstcatalyze a standard O-dealkylation to remove the 6′ methyl group(Guengerich, Chem. Res. Tox., 14:611 (2001)), producing thecarboxylmethyl ester of D-luciferin. Though a carboxyl ester ofluciferin would not be expected to react with luciferase to make light,it could be de-esterified by adding an esterase to the luciferindetection reagent used for a standard luminogenic P450 assay. Using thisassay strategy, the activity of CYP2D6 with the carboxyl ester ofluciferin-ME was found to be substantially increased over its activitywith luciferin-ME. The carboxyl ester of luciferin-ME also reactedstrongly with several other P450s including CYP2C19 (FIG. 20).

The carboxylmethyl ester of deoxyluciferin (luciferin-H) was screenedagainst the same P450 panel and showed substantial activity with CYP2C19and strong selectivity for this enzyme over the others tested (line 26in FIGS. 17-18).

The carboxylmethyl esters of luciferin-6-methyl ether and luciferin-Hwere good substrates for luminogenic P450 assays that include ade-esterification step after the P450 incubation (lines 26 and 33 inFIGS. 17-18). However, both compounds had poor solubility in the aqueousP450 assay buffer. To improve solubility, additional esters ofluciferin-6-methyl ether and luciferin-H were prepared. Picolinyl estersshowed improved aqueous solubility and a more limited pattern of P450cross reactivity (lines 27 and 32 in FIGS. 17-18). The carboxylpicolinyl ester of luciferin-6-methyl ether showed a preference for 2D6,1A1 and 1A2 while the carboxyl picolinyl ester of luciferin-H showedpreference for 2C19, 1A1 and 1A2. Both of these compounds had theunexpected property that treatment with an esterase was not required toachieve maximal signals. That is, a substantial difference in signal wasnot observed when comparing these assays plus and minus esterase.

These results suggest that the substrates may be oxidized by P450 on twosites: the 6′ site, as previously suggested, and the methylene of thepicolinyl group. A hydroxylation at the picolinyl methylene would makean unstable compound that would decay to form picolinyl aldehyde andD-luciferin. The sequential oxidation of a given substrate on multiplesites by a single P450 has been described (Cali et al., J. Biol. Chem.,266:7774 (1991)). Alternatively, if there were sufficient carboxylesterases present in the P450 preparation, these could account for theremoval of the picolinyl ester group. Experiments withluciferin-6-methylether picolinyl ester or luciferin-H picolinyl ester,with purified recombinant CYP2D6 or CYP2C19 preparations that weresubstantially free of carboxylesterases, clarified the roles of P450 andesterase to remove the picolinyl esters. When 1 unit of porcine esterasewas added to the purified P450 reactions a large signal was generated inthe 2 step assay format with the thermostable luciferase. In contrast,when the esterase was not added, less than 10% of the plus esterasesignal was observed. This relatively lower signal was neverthelesshigher than signals from reactions that lacked P450 activity altogether(when the essential P450 co-factor NADPH was withheld). Taken together,these results indicate that P450s (CYP2D6 and CYP2C19 in this example)modify the 6′ position of the 6′ methyl ether and 6′ deoxyluciferinpicolinyl esters of D-luciferin by hydroxylation or dealkylation,respectively. A second minor reaction, oxidation at the picolinylmethylene is also catalyzed by P450 leading to de-esterification.However, the major de-esterification observed with the recombinant P450sin insect cell membranes (Supersomes™) was catalyzed by non-P450carboxyl esterase activity present in the membrane preparation. Thelatter point was confirmed by HPLC analysis of the Supersome™ reactionswhere accumulation was observed of picolinyl alcohol, the expectedproduct of a carboxyl esterase reaction, whereas picolinyl aldehyde, theexpected product of a P450 mediate de-esterification, was not observed(data not shown).

Ethylene glycol esters of luciferin-6-methyl ether and luciferin-H werealso prepared. These were reactive with P450s and had greater solubilityin aqueous buffer than the methyl and picolinyl esters (lines 24-25 and36 in FIGS. 17-18). Unlike the picolinyl esters, the ethylene glycolesters required the addition of esterase to achieve maximum signals.Without added esterase diminished signals were observed (lines 24-25 inFIGS. 17-18).

D-luciferin esters were substrates for carboxylesterases in a two stepluminescent assay format similar to that used for P450s. D-luciferinmethyl ester, D-luciferin ethyl ester and D-luciferin picolinyl esterwere effective in this format. This was first suggested by results fromtwo of these compounds when screened against the panel of 21 recombinanthuman P450s. In those experiments, the P450s typically did not givesubstantially brighter signals than those observed with the controlreactions where the recombinant P450 membrane fractions were replacedwith a control membrane fraction that lacked P450. Though signals variedbetween P450 preparations no P450 preparation was much brighter than thecontrol. It seems likely then that the signals were not due to P450activity in the preparations but rather, variable amounts of carboxylesterase activities. Methyl, ethyl and picolinyl esters of D-luciferinwere also characterized as substrates for a purified preparation ofporcine esterase without any P450. In this format each ester linkedgroup was cleaved by the esterase to yield D-luciferin that in turnreacted with luciferase to make light.

There were some compounds that showed substantial light generatingactivity with aminoluciferin despite the fact that modifications weremade to the 6′ amine. This was unanticipated because D-luciferins with6′ modifications are typically substantially impaired as lightgenerating substrates for luciferase. A thermostable luciferase reactionwith 100 μM dimethyl amino luciferin was only about half as bright as areaction with 100 μM amino luciferin. This general phenomenon was alsotrue with N,N-benzyl-methyl-aminoluciferin,N,N-benzyl-ethyl-aminoluciferin and isopropyl amino luciferin. Othercompounds such as N,N bisbenzyl amino luciferin that react withluciferase to produce substantial amounts of light without priorreaction with a non-luciferase enzyme may also be good substrates forluciferase. Aminoluciferin derivatives that are good substrates forluciferase on their own can be used as scaffolds for furtherderivatization in the interest of making substrates for nonluciferaseenzymes for the luminogenic style of reactions described herein. In thiscase the luminogenic leaving group would be an aminoluciferin derivativesuch as dimethylaminoluciferin.

Thus, some luciferin derivatives with an ester group are useful ascarboxyl esterase substrates and those derivatives are not necessarilysubstrates for P450 enzymes. Other luciferin derivatives with an estergroup and a P450 substrate are useful as P450 substrates including thosewhere the corresponding derivative which lacks the ester group is not asubstrate, or a poor substrate, of at least some P450 enzymes. Moreover,for some luciferin derivatives, e.g., picolinyl luciferin-ME andpicolinyl luciferin-H, an exogenous esterase may not be needed to detectP450 activity in samples with endogenous esterase activity.

D. CYP3A Induction Assay in Human Hepatocytes

Certain compounds induce transcription of CYP3A gene expressionresulting in increased levels of CYP3A enzyme activity. It is thereforedesirable to have probes for detecting such inductions at the level ofP450 enzyme activity in cells such as hepatocytes (Madan et al., DrugMetab. Dispos., 31:421 (2003)). 6-(2,3,4,5,6pentafluorobenzyloxy)-luciferin and 6-phenylpiperizinexylyl-luciferinwere tested as probes for detecting CYP3A induction in hepatocytes(FIGS. 51-52). The results from screening a panel of P450 enzymesrepresenting most of those expressed in hepatocytes suggested that thesesubstrates would only react to a substantial extent with CYP3A enzymes(FIGS. 17-18).

Rifampicin is a prototypical CYP3A4 gene inducer and was used todetermine whether luminogenic P450 substrates could be used to detectinduction in a non-disruptive cell-based assay of human hepatocytes. Theluminogenic P450 substrates 6-(2,3,4,5,6 pentafluorobenzyloxy)-luciferinand 6-phenylpiperizinexylyl-luciferin were added to the medium ofcultured hepatocytes with the expectation that they would enter cellsand react with endogenous P450s to produce D-luciferin. The D-luciferinwas in turn expected to exit cells and accumulate in the culture medium.The medium was then sampled and combined with a luciferase reactionmixture to generate light in proportion to the amount of D-luciferinproduced by CYP3A enzymes.

Human hepatocytes were used to test the proposed CYP3A4 cell based assayapproach (FIGS. 51-52). Freshly plated human hepatocytes oncollagen-coated 96 well plates were obtained from Cambrex Bio Science(Walkerville, Md.). Cells were from a male donor and provided alive atnear confluence in 96-well plates at ambient temperature. Upon arrival,a plastic adhesive seal was removed from plates and the cells wereincubated in a 5% CO₂, 37° C. incubator for 2 hours in the shippingmedia. After 2 hours, shipping media was removed and replaced with 0.1mL per well Hepatocyte Culture Medium (HCM, Cambrex) pre-warmed to 37°C. Plates were returned to the incubator for 1 hour. After 1 hour, anadditional 0.1 mL cold HCM that had been supplemented with 0.25 mg/mLMATRIGEL® (BD Biosciences, Bedford, Mass.) was added to each well on topof the existing 0.1 mL HCM. Plates were then returned to the incubatorovernight. The next day culture medium was removed and replaced with 0.1mL fresh medium without MATRIGEL® that included 10 μM rifampicin (theCYP3A inducing compound) or vehicle alone (0.02% DMSO). Rifampicin andvehicle containing medium was changed the next day so that cells wereexposed to test compound for a total of 2 days. Vehicle controls wereused for measuring basal CYP450 activity. After 2 days of exposure torifampicin or vehicle medium was removed and replaced with 0.1 mL HCMsupplemented with 50 μM of 6-(2,3,4,5,6 pentafluorobenzyloxy)-luciferinor 6-phenylpiperizinexylyl-luciferin or these substrates plus the CYP3A4inhibitor ketoconazole. For background luminescence determinations, eachluminogenic substrate was added to a set of four empty wells (no cells).For basal CYP450 activity measurements, each luminogenic substrate wasadded to a set of four vehicle control wells. Forrifampicin-treated/induced CYP450 activity measurements, eachluminogenic substrate was added to four wells treated with the CYP3A4inducer, rifampicin. For inhibition of basal or rifampicin-inducedCYP450 activity measurements, each luminogenic substrate plus the CYP3A4inhibitor ketoconazole was added to four wells treated with vehiclealone and to four wells treated with the CYP3A4 inducer, rifampicin.

Samples were then incubated for 4 hours with the luminogenic substratesor luminogenic substrate plus inhibitor. At the end of the 4 hourincubation period with luminogenic substrates, 0.1 mL of medium wasremoved from each well to a 96 well opaque white luminometer plate atroom temperature and combined with 0.1 mL of a luciferin detectionreagent (from Promega Corporation). Luminescence was read on a platereading luminometer (VERITAS™, Turner BioSystems) 20 minutes aftercombining medium and luciferin detection reagent. The averageluminescence values of background wells were subtracted fromrifampicin-treated and untreated (vehicle control) values to give netCYP3A-dependent luminescence.

A basal level of luminescence was found in the vehicle control wells,apparently reflecting basal CYP3A activity and the induction of thisactivity by rifampicin was reflected as an increase in luminescence overcontrols with both substrates (FIGS. 51-52). The inhibition byketoconazole of both basal and rifampicin-induced activity with bothluminogenic substrates by the CYP3A4 inhibitor is consistent with theinterpretation that the basal and induced luminescence is a consequencea CYP3A reaction with luminogenic substrates. The fold inductionmeasured with 6-(2,3,4,5,6 pentafluorobenzyloxy)-luciferin was greaterthan that measured with 6-phenylpiperizinexylyl-luciferin. This may bedue to improved selectivity of the former substrate forrifampicin-inducible P450s or to better access to the P450s in thehepatocytes. The results demonstrate that both substrates are usefulprobes for detecting CYP3A gene induction at the level of P450 enzymeactivity. The approach has the advantage of leaving the hepatocytesintact so they could be subjected to additional analysis, for example,to test for cytotoxicity of a test compound with a cell viability assay.

Example 6 Luciferase Substrates

A. Firefly Luciferase

Firefly luciferase was shown to utilize luciferin derivatives that weremodified such that they formed ethers at rates comparable to the ratewith which luciferase uses natural luciferin as a substrate. Suchcompounds were not known to be able to be utilized by luciferase and infact one of the compounds was reported to not be a substrate for theenzyme. In addition, the data indicated that the utilization of suchderivatives can lead to: 1) inactivation of the luciferase, and 2)fluorescent labeling of the luciferase, but that certain compounds canincrease light production.

Materials and Methods

Gel and HPLC stop solution. Solid EDTA (free acid) was placed in abeaker with water and the pH of the solution adjusted to pH 7.0. Thesolution was then adjusted in volume to produce a solution 400 mM inEDTA.

Enzyme stop solution. A solution was prepared containing 20 mM Bis Tris,pH 6.5, 2 mM EDTA and 1 mg/ml bovine serum albumin.

Reaction buffer (2×). A solution was made containing 100 mM HEPESbuffer, pH 8.0; 10 mM MgCl₂.

DTT stock. Solid DTT (dithiothreatol) was dissolved in water and thenadjusted to pH 8.0 with solid sodium hydroxide. The solution was thenadjusted in volume to produce a solution 1 M in DTT.

Results

TABLE 37 Reaction Buffer 1M 20 mM Gel/HPLC Reaction (2X) DTT Coenzyme AStop Sol'n 10 mM ATP Luciferin 1 500 μl 0 μl 0 μl 0 μl 10 μl 10 μl 5 mMLuciferin BE 2 500 μl 0 μl 50 μl  0 μl 10 μl 10 μl 5 mM Luciferin BE 3500 μl 100 μl  0 μl 0 μl 10 μl 10 μl 5 mM Luciferin BE 4 500 μl 0 μl 0μl 100 μl  10 μl 10 μl 5 mM Luciferin BE 5 500 μl 0 μl 0 μl 0 μl  0 μl10 μl 5 mM Luciferin Be 6 500 μl 0 μl 0 μl 0 μl 10 μl 10 μl 5 mMLuciferin ME 7 500 μl 0 μl 50 μl  0 μl 10 μl 10 μl 5 mM Luciferin ME 8500 μl 100 μl  0 μl 0 μl 10 μl 10 μl 5 mM Luciferin ME 9 500 μl 0 μl 0μl 100 μl  10 μl 10 μl 5 mM Luciferin ME 10 500 μl 0 μl 0 μl 0 μl  0 μl10 μl 5 mM Luciferin Me

After producing the solutions in Table 37, the volume of all reactionswas adjusted to 990 μl with water. Reactions were started by addition of10 μl of QUANTILUM® Luciferase (Promega Corp.; 14.9 mg/ml) to areaction, mixing, removing 100 μl to a luminometer tube and reading thelight produced by the reaction. While the light reading was being taken,a 10 μl sample of the reaction was diluted to 500 μl with enzyme stopsolution (cooled on ice to 4° C.) mixed and placed on ice and a secondsample of 90 μl of reaction was added to 10 μl of Gel/HPLC stop and thetube placed at −20° C. A timer was started with the start of the firstreaction and the start times of the remaining reactions was noted. At15, 30, 45 and 60 minutes, each reaction was sampled as above and thelight produced in the luminometer tubes was measured. At 7 and 22minutes, the light produced in the luminometer tubes was read, but thereactions were not sampled.

After all samples were taken, 50 μl of the sample diluted into enzymestop solution was mixed with 50 μl of Steady Glo Luciferase Reagent(Promega) and the light produced measured after incubating at roomtemperature for 10 minutes. This measured the active luciferase in theinitial reaction.

Samples of the materials given Gel/HPLC stop solution and frozen werethawed and analyzed by HPLC in addition, selected samples were mixedwith SDS PAGE loading buffer and fractionated on 4-20% SDS PAGE gels andvisualized on a Typhoon Phosphoimager.

Results

The following readings for light emission from the reactions wereobtained (Table 38).

TABLE 38 Light readingat time Reaction Substrate Addition 0 min 7 min 15min 22 min 30 min 45 min 60 min 1 Luc'in No 135.2 22.61 12.79 9.2566.928 4.879 3.311 BE addition 2 + Co A 139.5 11.92 10.99 9.941 7.3797.284 6.433 3 + DTT 948 532.8 417 369.5 331.6 273.5 226.9 4 + EDTA 1.0040.771 0.639 0.562 0.461 0.425 0.354 5 w/o ATP 0.015 0 0.008 0.005 0 00.01 6 Luc'in No 23.46 3.144 1.767 1.317 1.22 0.982 0.851 ME addition7 + Co A 18.31 2.276 1.239 1.178 1.145 0.916 0.714 8 + DTT 38.69 6.424.073 3.31 2.708 2.202 1.996 9 + EDTA 1.067 0.117 0.058 0.042 0.0240.046 0.012 10 w/o ATP 0 0.017 0.016 0 0 0.017 0.011

Analysis of the samples by HPLC indicated that at least 20% of theluciferin derivative was utilized by the time the 15 minute sample wastaken except in the reactions where no ATP was added or where EDTA waspresent in the reaction solution. In those reactions, very littleutilization of the derivative was seen under these conditions.

The following readings for light emission from the active enzyme in thesamples were obtained (Table 39).

TABLE 39 Light reading from residual active enzyme at Reac- Sub- timetion strate Addition 0 min 15 min 30 min 45 min 60 min 1 Luc'in No 129.769.9 47.31 37.94 29.08 BE addition 2 + Co A 186.7 179.6 179.4 157.9161.8 3 + DTT 194 233.7 224.7 219.5 226.4 4 + EDTA 170 142.7 140.4 141.7135.4 5 w/o 168.3 174.1 171.7 172.9 164 ATP 6 Luc'in No 141.7 35.4 91.8285.72 81.76 ME addition 7 + Co A 163.3 161.8 147.7 144.7 145.6 8 + DTT170 172.3 164.3 161.6 167.6 9 + EDTA 150.5 150 152.1 152.8 151 10 w/o170 168.9 175.6 181.9 173.6 ATP

As seen from the data in the residual active enzyme table above, littlechange was seen in the residual enzyme levels in the reactions aboveexcept for the reactions without additive. The reactions withoutadditive show a time dependent drop with the magnitude of the dropdependent upon the derivative present in the reaction.

SDS-PAGE analysis of samples of these reactions followed byvisualization of the image of the gel using a Typhoon imagerdemonstrated that the protein in reactions given no addition did becomefluorescently labeled in a time dependent manner.

Thus, substantial and time dependent depletion of the luciferinderivative was seen under conditions where luciferin is highly active(reactions above with no addition, or coenzyme A or DTT addition), lightproduction was seen in reactions with the derivative, and enzymeinactivation and protein labeling were seen in these reactions undersome conditions. Accordingly, this enzyme can utilize these luciferinderivatives. As mentioned above, derivatives where the oxygen ofluciferin is in an ether linkage had been reported in the literature notto be substrates for luciferin, and thus the fact that these materialsare effective substrates is surprising.

After producing the solutions in Table 40, the volume of all reactionswas adjusted to 990 μl with water. Reactions were started by addition of10 μl of QUANTILUM® Luciferase (Promega Corp.; 14.9 mg/ml) to areaction, mixing, removing 100 μl to a luminometer tube and reading thelight produced by the reaction. While the light reading was being taken,a 10 μl sample of the reaction was diluted to 500 μl with enzyme stopsolution (cooled on ice to 4° C.) mixed and placed on ice and a secondsample of 90 μl of reaction was added to 10 μl of Gel/HPLC stop and thetube placed at −20° C. A timer was started with the start of the firstreaction and the start times of the remaining reactions was noted. At15, 30, 45 and 60 minutes, each reaction was sampled as above and thelight produced in the luminometer tubes was measured. At 7 and 22minutes, the light produced in the luminometer tubes was read, but thereactions were not sampled.

TABLE 40 20 mM Reaction Coenzyme 10 mM Reaction Buffer (2X) 1M DTT A ATPLuciferin 3A 500 μl 0 μl 0 μl 10 μl 10 μl 5 mM Luciferin 3B 500 μl 0 μl50 μl  10 μl 10 μl 5 mM Luciferin 3C 500 μl 100 μl  0 μl 10 μl 10 μl 5mM Luciferin 3D 500 μl 0 μl 0 μl  0 μl 10 μl 5 mM Luciferin 4A 500 μl 0μl 0 μl 10 μl 10 μl 5 mM 2OHETox Luciferin 4B 500 μl 0 μl 50 μl  10 μl10 μl 5 mM 2OHETox Luciferin 4C 500 μl 100 μl  0 μl 10 μl 10 μl 5 mM2OHETox Luciferin 4D 500 μl 0 μl 0 μl  0 μl 10 μl 5 mM 2OHETox Luciferin

The following light readings were collected on a Turner 20/20 (Tables 41and 42).

TABLE 41 Native Luciferin Data Time Rx Rx. Type 0 7 15 22 30 45 60 3A Nooff scale off 1,920,967,552 1,340,545,280 1,080,656,384 698,897,920507,915,488 Additive scale 3B + Co A off scale off 1,817,884,6721,207,508,736 1,075,446,272 727,134,016 516,780,608 scale 3C + DTT offscale off off scale 1,828,845,952 1,369,086,080 882,840,768 640,560,896scale 3D no ATP 1,013,266 7,266 2,253 248,151 194,980 27,586 22,270

TABLE 42 2 Hydroxy EthoxyEther Luciferin Rx Rx. Type 0 7 15 22 30 45 604A No 749,597 114,541 63,691 259,945 233,600 51,233 44,782 Additive 4B +Co A 810,864 125,718 61,287 326,424 169,972 78,909 45,156 4C + DTT2,515,513 294,885 146,849 335,031 237,487 67,064 4D no ATP 87 106387,497 218,745 11,499 20,100

The HPLC profiles from the reactions indicated that the peakrepresenting the unreacted luciferin substrates in these reactions wasdeclining at a very similar rate except for the reactions not given ATP.This rate was very similar to the rate seen for utilization of LuciferinBE and Luciferin ME.

The following readings for light emission from the active enzyme in thesamples were obtained (Table 43).

TABLE 43 Sample Time [min] Reaction 0 15 30 45 60 3A 183.6 170.7 175.6164.6 146.5 3B 243.6 218.2 211.1 208.2 192.1 3C 188.7 203.9 187.9 191.5188.7 3D 199.9 194.2 190.4 197.4 194.2 4A 153.2 124.8 111.6 108.4 102.54B 207.7 202.5 172.8 191.3 180.8 4C 185.6 190.8 188.1 199.7 190.6 4D174.3 178.3 176.2 182.2 192

While the generation of light from transformation of native luciferin byQUANTILUM® luciferase does not lead to a large loss of luciferaseactivity (see reaction 3A in Table 43), the transformation of the 2hydroxyethoxyluciferin derivative led to significant and substantialreduction in enzyme activity (see reaction 4A above). This can beprevented to a very large extent by the presence of various compounds,such as DTT (see reaction 4C above). While there have been reports thatthe rapid turnover of luciferin by the QUANTILUM® enzyme can lead tosome enzyme inactivation, the inactivation rate for reactions usingluciferin derivatives appears to be much more rapid. In addition, theinclusion of DTT to reactions using oxygen ether derivatives ofluciferin with QUANTILUM® enzyme increased the light production.

SDS PAGE analysis of the samples indicated that a faint fluorescentprotein band was formed in reactions 3A and 4A but that there was noband formed under the other reaction conditions.

Since the HPLC profiles of these reactions showed about an equal rate ofsubstrate disappearance for the luciferin derivatives as for nativeluciferin, the rate for the modified substrates and luciferin areapproximately the same. Since the light production for the reactionswith the derivative is far lower than for native luciferin, turnover ofthese derivatives by QUANTILUM® luciferase does not result in asefficient light production as with native luciferin.

B. Scaffolds

In this example, an experiment was performed that demonstrates thatlight production by some luciferin derivatives may be dependent upon theparticular chemical groups attached to the luciferin. However, onegeneral observation that can be made is that aminoluciferin derivativesproduce far more light than oxyethers of luciferin. Thus, theaminoluciferin backbone can be used as a scaffold to prepare otherluciferin derivatives that are substrates of luciferase or derivativeswhich are substrates for a nonluciferase enzyme and prosubstrates ofluciferase that, after reacting with the nonluciferase enzyme, yield aproduct that is an aminoluciferin derivative, e.g., dimethylaminoluciferin or isopropylaminoluciferin.

Materials and Methods

The DTT, Coenzyme A, and QUANTILUM® stock solutions are the same asthose described herein.

An enzyme addition solution was created that contained 400 mM HEPES, pH8.0, 40 mM MgCl₂, 2000 μg/ml BSA, 400 μM ATP and 8 μl of QUANTILUM®Luciferase (14.9 mg/ml). This solution was diluted 1:1 with water toproduce an enzyme solution labeled “No addition.” This solution wasdiluted 1:1 with 400 mM DTT, pH 8.0 to produce a solution labeled+DTT.This solution was diluted 1:1 with 4 mM Coenzyme A to produce a solutionlabeled+Co A. Finally, this solution was diluted 1:1 with a solutionhaving both 4 mM Coenzyme A and 400 mM DTT to produce a solutionlabeled+Co A & DTT.

Results

The various luciferin derivatives listed below were diluted from 5 mMstock solutions to produce solutions at 200, 100, 50, 25, 12.5, 6.25 and3.125 μM derivative. 50 μl samples of these solutions were placed in thewells of luminometer microtiter plates, row A to row G, respectively.Row H of the plates was given 50 μl of water.

Reactions in these plates were initiated by addition of 50 μl ofsolution containing QUANTILUM® luciferase using a 12 channelmicropipette. The solutions added to the rows were: columns 1, 5, and9—no addition solution; columns 2, 6, and 10—+Co A solution; columns 3,7, and 11—+DTT solution, and; columns 4, 8, and 12—+Co A and DTTsolution. After addition of the enzyme solution to the first row onevery plate, a timer was started. After addition of the enzyme solutionto the last row on each plate, the plate was sealed and read on aVERITAS™ luminometer. The following light emission values (Tables 44-46)were obtained (“alone” indicates no addition).

TABLE 44 Plate 1 dimethylamino luciferin diethylaminoluciferinDerivative Co A + Co A + Conc. Alone Co A DTT DTT Alone Co A DTT DTT100    27421864 27183208 12461676 10059717 9082149 10142081 21809831960750 50    25967878 24213794 12561822 10272291 8176569 72061571826771 1538668 25    22309938 20845376 11892757 9925327 6763022 56238861304383 1067954 12.5  17014016 15077778 9239960 6714299 5418990 4485779682672 645536 6.25 9091842 9170130 5998175 2602679 4009797 3457051165732 261211 3.13 4526705 4109624 2766839 260502 2259570 1941060 4772793423 1.07 304706 208370 702319 7119 656110 688887 5192 20592 0   786756 1135 1572 1154 1188 1802 4723 methylbenzylaminoluciferin Plate 1Derivative Co A + Conc. Alone Co A DTT DTT 100    85579872 8911778450806656 48409604 50    73867336 78390592 54238960 47965140 25   61572912 63598896 50632712 42613860 12.5  50528576 50418220 3666361230367190 6.25 43052788 35730272 21756222 19738584 3.13 34516960 235012828088743 9404429 1.07 25907602 12863007 929977 2037020 0   10614 83784621 2959

TABLE 45 Plate 2 MAO #3 GST #4 GST #3 Derivative Co A + Co A + Co A +Conc. Alone Co A DTT DTT Alone Co A DTT DTT Alone Co A DTT DTT 100348013 741882 994617 1019425 2119 1853 1618 1990 16388 18343 21472 1912950 179585 444494 868903 865209 1598 1366 1354 1729 17409 15440 2288820009 25 272608 588881 733675 701503 1413 1026 1492 1640 15957 1479121256 18145 12.5 252130 467021 525765 509896 1692 1275 1474 1891 1175210674 16587 14298 6.25 201597 321914 348398 82531 1571 1562 1633 17307682 7149 11164 10423 3.13 131633 176814 200074 171233 1537 1635 14582034 4913 4832 9106 8826 1.07 94583 122510 111682 52637 1146 1533 11021714 3232 3081 5953 6964 0 528 450 734 1077 488 463 684 940 450 373 6871299

TABLE 46 2hydroxyethoxyluciferin ether Luciferin BE Luciferin ME Co A +Co A + Co A + Plate 3 Alone Co A DTT DTT Alone Co A DTT DTT Alone Co ADTT DTT 100 53804 52640 128249 94724 31581 33199 54592 64460 3589 33895399 4550 50 38444 39320 98407 87170 30609 38349 80001 98716 2690 27044052 3635 25 27356 28679 76612 67303 40093 53097 84034 90306 2083 22243491 2992 12.5 19586 21636 61882 54630 40486 57996 94861 97371 1563 17682961 2510 6.25 14686 18532 48458 42815 38838 58288 100044 105912 12401673 2844 2882 3.13 10769 14986 36967 32555 32994 55778 86445 80626 10531360 3332 2853 1.07 8807 13847 24491 22105 29234 50144 57066 51799 11971523 3160 3180 0 462 852 952 1428 856 567 1294 1495 753 721 1378 1275

An observation made when gathering this data was that the presence ofDTT at high concentrations decreased light production from aminoderivatives of luciferin while DTT increased light production fromoxyether analogs. Thus, these data clearly show that light productioncan vary up to at least 35,000 fold depending upon the particularconcentration of the derivative, and up to at least 12,000 folddepending on the derivative chosen.

B. Thermostable Luciferases

In this example, a second luciferase was demonstrated to utilizeluciferin derivatives. Again, the transformation of these substrates ledto covalent modification of the protein in such a way that it becomesfluorescently tagged.

Materials and Methods

A thermostable luciferase (see U.S. Pat. No. 6,602,677; Luc 146-1H2) wasobtained in pure form at a purified stock protein concentration of 6.8mg/ml. This stock was used to produce enzyme stock solution having of200 μg/ml enzyme solution in 1 mg/ml BSA, 200 mM HEPES buffer, pH 8.0,20 mM MgCl₂.

Luciferin BE and Luciferin CEE (Promega Corp.) were diluted with waterfrom an initial concentration of 5 mM to solutions of 400, 80 and 16 μM.

Solutions were made using a 1 M DTT solution, pH adjusted to 8.0, a 20mM Coenzyme A solution in water (both described in examples above), a100 mM ATP solution and water to create the following solutions: Sol'A,100 μM ATP; Sol'B, 100 μM ATP, 4 mM Coenzyme A; Sol'n C, 100 μM ATP, 400mM DTT, and; Sol'n D, 100 μM ATP, 4 mM Coenzyme A and 400 mM DTT.

The following reactions were prepared (Table 47)

TABLE 47 Luciferin Reaction stock, amt. Water Sol'A Sol'B Sol'C Sol'D 125 μl 25 μl 25 μl  0 μl 0 μl 0 μl Luciferin BE, 400 uM 2 25 μl 25 μl 0μl 25 μl  0 μl 0 μl Luciferin BE, 400 μM 3 25 ul 25 μl 0 μl 0 μl 25 μl 0 μl Luciferin BE, 400 μM 4 25 ul 25 μl 0 μl 0 μl 0 μl 0 μl LuciferinBE, 400 μM 5 25 μl 25 μl 25 μl  0 μl 0 μl 0 μl Luciferin BE, 20 μM 6 25μl 25 μl 0 μl 25 μl  0 μl 0 μl Luciferin BE, 20 μM 7 25 μl 25 μl 0 μl 0μl 25 μl  0 μl Luciferin BE, 20 μM 8 25 μl 25 μl 0 μl 0 μl 0 μl 0 μlLuciferin BE, 20 μM 9 25 μl 25 μl 25 μl  0 μl 0 μl 0 μl Luciferin BE, 16μM 10 25 μl 25 μl 0 μl 25 μl  0 μl 0 μl Luciferin BE, 16 μM 11 25 μl 25μl 0 μl 0 μl 25 μl  0 μl Luciferin BE, 16 μM 12 25 μl 25 μl 0 μl 0 μl 0μl 0 μl Luciferin BE, 16 μM 13 25 μl 25 μl 25 ul  0 μl 0 μl 0 μlLuciferin CEE, 400 μM 14 25 μl 25 μl 0 μl 25 μl  0 μl 0 μl LuciferinCEE, 400 uM 15 25 μl 25 μl  0 μl 0 μl 25 μl  0 μl Luciferin CEE, 400 μM16 25 μl 25 μl 0 μl 0 μl 0 μl 0 μl Luciferin CEE, 400 μM 17 25 μl 25 μl25 μl  0 μl 0 μl 0 μl Luciferin CEE, 80 μM 18 25 μl 25 μl 0 μl 25 μl  0μl 0 μl Luciferin CEE, 80 μM 19 25 μl 25 μl 0 μl 0 μl 25 μl  0 μlLuciferin CEE, 80 μM 20 25 μl 25 μl 0 μl 0 μl 0 μl 0 μl Luciferin CEE,80 μM 21 25 μl 25 μl 25 μl  0 μl 0 μl 0 μl Luciferin CEE, 16 μM 22 25 μl25 μl 0 μl 25 μl  0 μl 0 μl Luciferin CEE, 16 μM 23 25 μl 25 μl 0 μl 0μl 25 μl  0 μl Luciferin CEE, 16 μM 24 25 μl 25 μl 0 μl 0 μl 0 μl 0 μlLuciferin CEE, 16 μM 25 0 μl 50 μl 25 μl  0 μl 0 μl 0 μlResults

After assembly, the reactions were initiated by addition of 25 μl of thethermostable luciferase stock solution above and mixing the tubecontents. A 50 μl sample was immediately removed and placed in aluminometer tube and the light read and the tube then replaced in a tuberack at room temperature. While the light reading was being taken, a 5μl sample of the remaining reaction (which was kept at room temperature)was diluted into 495 μl of 50 mM BisTris, pH 6.5, 1 mg/ml BSA, 1 mM EDTAprechilled to 4° C. and the tube mixed and placed on ice. The lightproduced by the samples in the luminometer tubes were re-read at 20, 40,60, 90 and 120 minutes post enzyme addition. Additional 5 μl sampleswere taken and diluted as above at 60 and 120 minutes post enzymeaddition. Once all these readings and samples were taken, 50 μl of thediluted samples was mixed with 50 μl of Steady Glo in luminometer tubes,the tubes incubated 10 minutes on ice then the light produced was readon a Turner TD 20/20 luminometer.

The following light readings from the reaction samples directly placedinto luminometer tubes were obtained (Table 48).

TABLE 48 Time [min] Reaction Substrate Additive 0 20 40 60 90 120 1L'in-BE 100 Alone 160 48.21 19.25 8.305 2.843 1.311 2 + Co A 191.9 63.8529.28 14.78 6.652 3.014 3 + DTT 148.7 77.9 48.39 32.26 21.71 14.25 4 +Co A & 140.4 71.5 45.66 29.32 19.92 13.08 DTT 5 L'in-BE 25 Alone 623.273.96 28.76 13.42 5.528 2.549 6 + Co A 972.4 127.4 58.9 31.96 17.549.727 7 + DTT 448.6 84.21 43.77 27.21 18.48 12.93 8 + Co A & 402 85.3745.87 27.94 18.94 13.13 DTT 9 L'in-BE Alone 785.5 62.64 22.11 11.225.189 2.995 6.26 10 + Co A 2575 207.1 83.73 45.54 24 14.42 11 + DTT455.6 71.9 33.73 21.49 13.67 9.847 12 + Co A & 436.9 80.41 38.47 22.8316.08 12.01 DTT 13 L'in-CEE Alone 20.46 1.18 0.868 0.788 0.627 0.544 10014 + Co A 97.88 10.3 7.127 5.562 3.969 2.845 15 + DTT 22.47 2.233 1.2921.164 1.093 0.958 16 + Co A & 25.41 3.278 2.027 1.666 1.27 1.157 DTT 17L'in-CEE Alone 59.87 3.176 1.919 1.305 1.178 1.03 25 18 + Co A 489.245.45 25.08 16.96 11.03 7.309 19 + DTT 56.59 3.761 2.196 1.668 1.2741.157 20 + Co A & 57.98 5.721 3.272 2.192 1.379 1.174 DTT 21 L'in-CEEAlone 121 5.884 3.316 2.352 1.569 1.154 6.25 22 + Co A 1829 114 57.8237.53 24.66 17.02 23 + DTT 72.52 5.903 3.073 2.089 1.431 1.125 24 + Co A& 77.35 7.958 3.656 2.312 1.414 1.134 DTT 25 No L'in ATP Only 198.81.177 0.123 0.119 0.101 0.089 Con.

The table below (Table 49) contains the light values obtained uponaddition of the diluted reaction samples into Steady Glo reagent.

TABLE 49 Time Sample Taken Reaction Substrate Additive 0 min 60 min 120min 1 L'in-BE 100 Alone 1245 156.4 61.75 2 + Co A 1486 242.2 82.03 3 +DTT 1762 952.1 691 4 + Co A & 1780 1016 778.7 DTT 5 L'in-BE 25 Alone1309 157.1 72.57 6 + Co A 1610 277.8 143.2 7 + DTT 1675 910.2 738.9 8 +Co A & 1925 1102 836.3 DTT 9 L'in-BE Alone 2057 406.6 267.7 6.25 10 + CoA 1841 518.6 349.1 11 + DTT 1860 1173 1067 12 + Co A & 1943 1235 1163DTT 13 L'in-CEE Alone 1262 1203 1273 100 14 + Co A 1127 1055 770.5 15 +DTT 1168 501.5 1478 16 + Co A & 1745 1319 1343 DTT 17 L'in-CEE Alone1345 973.4 860.3 25 18 + Co A 1547 777.5 588.7 19 + DTT 1116 1372 139320 + Co A & 529.6 1268 1329 DTT 21 L'in-CEE Alone 1402 1158 1073 6.2522 + Co A 1647 976.8 921.4 23 + DTT 1631 1544 1695 24 + Co A & 1836 16251622 DTT 25 No L'in ATP Only 1415 1685 1863 Con.

These data indicate that the tested thermostable luciferase can utilizethese two luciferin derivatives as substrates. With these luciferinderivatives and the tested luciferase, DTT does not appear to have theability to dramatically stimulate light production as was seen with theQUANTILUM® enzyme. However, as seen with the QUANTILUM® Luciferase,utilization of these substrates by the thermostable luciferase resultedin an inactivation of the enzyme that can be reduced by the addition ofDTT to the reaction. The addition of coenzyme A increased the level oflight production, however, this compound appeared to have differentialeffects upon the retention of activity of the enzyme with the additivealone, somewhat decreasing the inactivation seen with luciferin BE andenhancing the inactivation with luciferin CEE.

Summary

Derivatives that were designed such that the phenolic hydroxyl group onluciferin was modified to be an oxygen ether or a derivatized aminesurprisingly were utilized by luciferase. For instance,N-(3-hydroxypropyl)-aminoluciferin (FIG. 7B) is a substrate forluciferase that provides sufficient light output for a luminescencereaction.

The fact that light production from the derivatives varied over severalorders of magnitude was also surprising. As described herein, recentdata clearly shows that, at least for QUANTILUM® luciferase, utilizationof the derivatives can be as rapid as that seen for native luciferin.However, light production can be over 10,000 fold less than fromD-luciferin, indicating that light production may not be an effectivemeans to determine if a derivative is utilized effectively. Thus, thelow level of light produced from many of the derivatives coulderroneously have been attributed to very low levels of contaminatingnative luciferin rather than to low level light production from therapid utilization of the actual derivatives.

For instance, N-(3-hydroxypropyl)-aminoluciferin (FIG. 7B) is asubstrate for luciferase that provides sufficient light output for aluminescence reaction.

Further work demonstrated that both the thermostable and QUANTILUM®luciferases were inactivated in the presence of some of the derivativesand that some compounds, such as DTT, may alter light production. Theeffect of DTT on light production can result in either no effect onlight production, an increase in light production, or a decrease inlight production, dependent upon the specific combination of luciferaseand derivative employed. Moreover, another compound, coenzyme A,substantially increased light production from a luciferin derivative,however, it may: 1) increase the rate of enzyme inactivation 2) have noapparent effect on enzyme inactivation, or 3) greatly decrease the rateof enzyme inactivation dependent upon the particular combination ofluciferase and derivative.

While thiol compounds such as DTT have been shown to preventinactivation of luciferase during the transformation of nativeluciferin, and that coenzyme A can stimulate light production, theresults with luciferin derivatives on the invention were surprisingbecause: the derivatives were unknown and it was unknown whether theywere substrates for luciferase, and the phenolic oxygen of luciferin wasthought to be crucial for luciferin to act as a substrate, and so atleast some of the derivatives would not be expected to be substrates. Ifthe derivatives were likely not to be substrates, there would be noreason to expect that their biotransformation by luciferase would leadto inactivation of the enzyme. Moreover, since the derivatives were notknown to inactivate the enzyme, there would be no expectation that DTTcould alter light production in reactions with certain derivatives.Therefore, there would be no reason to expect that any particularderivative could substantially increase light output from transformationof the derivative.

These observations suggest particular modifications that are bestavoided as they may be effectively utilized by luciferase withouttransformation, producing a substrate that can give a very highbackground signal. In addition, knowing that such a derivative may beutilized may identify why some derivatives produce unexpected lightsignals when used at different concentrations. Moreover, identifyingderivatives that can inactivate luciferase is useful in assaydevelopment. For example, for an assay that generates a steady lightsignal over an extended period of time, an agent that preventsinactivation of the enzyme by the unspent derivative that is in thereaction may be employed. Thus, a solution for the measurement of theamount of luciferin derivative transformed by an enzymatic or chemicalprocess that includes an agent to prevent the inactivation of luciferasein the solution is envisioned.

The observations described herein provide for a synthetic strategy. Thisstrategy is based upon the observation that untransformed derivativesproduce light and that different derivatives can produce far differentlight levels. Prior to these observations, it was likely that allsubstrates that would be useful for assay of an enzyme through thetransformation of a luciferin derivative would need to produce a nativeluciferin moiety. Understanding that light can be produced by luciferinsby transformation of a luciferin derivative indicated that the enzymatictransformation need not produce native luciferin to be able to bemeasured by light production, only that the production have a measurablydifferent level of light production (either higher or lower) than theoriginal derivative. This strategy simplifies potential substrate designand suggests that the substrates that will generate the strongest lightsignals without generating native luciferin are those that contain anamino group in place of the phenolic oxygen and that the amino group canbe a tertiary amine. If so, one of the attached groups likely is nolarger than a methyl group while the other can be as large as at least abenzyl group.

Example 7 Luciferase Reactions with Fluoroluciferins are Less pHSensitive

A. 5′-fluoroluciferin

As shown below, firefly luciferase (QUANTILUM® Luciferase, E170, PromegaCorporation, Madison, Wis.) utilized 5-fluoroluciferin (FIG. 24) in aless pH-dependent manner than it utilized luciferin.

Materials and Methods

QUANTILUM® luciferase was diluted to 14.7 ng/ml in Glo Lysis Buffer(part E266B, Promega Corporation) containing 1 mg/ml BSA (partBP1605-100, Fisher Scientific, N.J.).

To test the pH-dependence of the luciferase signal, the luciferasedetection reagents contain one of 3 buffers each at one of 4 pHs, andthis combination was identical for luciferin and 5′-fluoroluciferin. Theluciferase detection reagents were created using a stock solutioncontaining 0.1850 g of DTT (V3155, Promega Corporation), 0.0121 g ATP(part 27-1006-01, Amersham Biosciences, N.J.), 80 μl of 0.5 M CDTAstock, pH 8.0 (AA842, Promega Corporation), and 240 μM of 1 M magnesiumsulfate stock (AA319, Promega Corporation) in 24 ml of water. This wassplit into 2 aliquots and luciferin (E1602, Promega Corporation) or5-fluoroluciferin (Promega Biosciences Inc., CA) were added to finalconcentrations, in this solution, of 787.5 μM. Aliquots of thesesolutions were then placed into tubes and buffers were added to 80 mM.The final luciferin detection reagents therefore contained 80 mM buffer,30 mM DTT, 500 μM ATP, 1 mM CDTA, 6 mM magnesium sulfate and 630 μMluciferin or 5-fluoroluciferin.

The buffers were tricine at pH 8.327, 8.100, 7.883 and 7.673; HEPES atpH 7.892, 7.720, 7.521, and 7.303; and MOPS at pH 7.499, 7.296, 7.133,6.900.

Luminescent reactions were initiated by combining 100 μl of theluciferase-containing solution and 100 μl of the luciferase detectionreagents. Luminescence was integrated over 1 second per sample using theVERITAS™ plate luminometer (Turner BioSystems). N=3 for each result.

Results

The average luminescence measured for each set of samples is listed inTable 50 (relative standard deviation ≦4.0%). The relative luminescenceshown is the average luminescence of each triplicate divided by theaverage luminescence for the luciferin-based luciferase detectionreagent at pH 8.3.

TABLE 50 Absolute Relative Relative Absolute Luminescence, LuminescenceLuminescence Luminescence, 5- of Luciferin of 5-FluoroluciferinLuciferin Fluoroluciferin (% of 8.3 (% of 8.3 pH Buffer (RLU) (RLU)Luciferin) Luciferin) 8.327 Tricine 1,463,179 317,030 100% 22% 8.100Tricine 1,422,936 357,041 97% 24% 7.883 Tricine 1,254,769 357,883 86%24% 7.673 Tricine 1,041,115 351,411 71% 24% 7.892 HEPES 976,654 377,49867% 26% 7.720 HEPES 815,317 378,927 56% 27% 7.521 HEPES 661,829 360,30845% 25% 7.303 HEPES 482,253 323,456 33% 22% 7.499 MOPS 707,832 334,28848% 23% 7.296 MOPS 585,743 331,515 40% 23% 7.133 MOPS 490,814 325,33734% 22% 6.900 MOPS 330,277 272,127 23% 19%

These data show that the luminescence increased almost 5-fold over thepH range tested when the luciferase detection reagent containedluciferin, whereas the luminescence changed by only about 40% when theluciferase detection reagent contained 5′-fluoroluciferin. Furthermore,the luciferase detection reagent was less sensitive to the buffercomposition when it contained 5-fluoroluciferin than when it containedluciferin. The luciferase luminescence increased by 28% in pH 7.89reagent when the luciferin-containing buffer was changed from HEPES totricine, however, the 5′-fluoroluciferin containing reagent onlydecreased by 6% when these buffers were changed. The pH insensitivity ofthe luciferase detection reagent containing 5′-fluoroluciferin could bebeneficial if luciferase reactions are run at low pH, especially thosebelow approximately 6.9, or where pH shifts between different samplesmay occur (for example, if the sample to be measured is highly bufferedbut not all samples are the same pH). Therefore, the 5′-fluoroluciferinscaffold may be modified to include a site for nonluciferase enzymeactivity or chemical activity which derivative could be used to monitorluciferase activity, a nonluciferase enzyme activity, or a chemicalactivity.

B. Other fluoroluciferins

Materials and Methods

Reactions were assembled to compare the spectrum generated by5′-fluoroluciferin, 7′-fluoroluciferin and luciferin. Reagent wascreated containing 200 mM HEPES, 200 mM PIPES, 1 mM CDTA, 0.5% TergitolNP-9, 0.03% Benzyl dodecyldimethyl ammonium bromide, 30 mM thiourea, 3mM ATP, 6 mM magnesium sulfate, and 0.5 mM luciferin or fluoroluciferin.pH of the reagent was 7.4. All compounds except fluoroluciferin and ATPwere purchased from Sigma Chemical. ATP was purchased fromAmersham/Invitrogen. Luciferin and both fluoroluciferins were made atPromega Biosciences, Inc. Reactions were initiated by adding equalvolumes (0.5 ml) of reagent and DMEM medium (Gibco/Invitrogen)containing 0.1% Prionex® (Pentapharm) and 2.94×10⁻² mg/ml QUANTILUM®Luciferase (Promega Corporation).

Spectra were measured using 0.5s integration with 1 nm increments on aSpex Fluorolog.

For pH titrations, reagent was created containing 100 mM HEPES, 100 mMPIPES, 100 mM Tricine, 1 mM CDTA, 0.5% Tergitol NP-9, 0.03% Benzyldodecyldimethyl ammonium bromide, 30 mM thiourea, 3 mM ATP, 6 mMmagnesium sulfate, and 0.15 mM 7′-fluoroluciferin. All compounds exceptfluoroluciferin and ATP were purchased from Sigma Chemical. APT waspurchased from Amersham/Invitrogen. The pH of solution was adjustedwithin the range of 8.5 to 6.4. Reactions were initiated by adding equalvolumes (0.1 ml) of reagent and DMEM medium (Gibco/Invitrogen)containing 0.1% Prionex® (Pentapharm) and 1.4×10-5 mg/ml QUANTILUM®Luciferase (Promega Corporation).

Luminescence was measured for 0.5 seconds per well using TurnerBiosystems VERITAS™.

For ATP titrations for luciferin and 5′ fluoroluciferin, reagent wascreated containing 100 mM HEPES pH 7.5, 0.5 mM CDTA, 30 mM DTT, 6 mMmagnesium sulfate, and 0.5 mM 5′-fluoroluciferin or luciferin. Allcompounds except 5′-fluoroluciferin and DTT were purchased from SigmaChemical. To some of this reagent 5 mM ATP (Amersham/Invitrogen) wasadded, then serially diluted by half-logs to 0.05 mM. Reactions wereinitiated by adding equal volumes (0.1 ml) of reagent and DMEM medium(Gibco/Invitrogen) containing 0.1% Prionex® (Pentapharm) and 1.4×10-5mg/ml QUANTILUM® Luciferase (Promega Corporation).

For ATP titrations for 7′-fluoroluciferin, reagent was createdcontaining 200 mM HEPES, 200 mM PIPES, 1 mM CDTA, 0.5% Tergitol NP-9,0.03% Benzyl dodecyldimethyl ammonium bromide, 30 mM thiourea, 10 mMmagnesium sulfate, and 0.15 mM 7′-fluoroluciferin. This solution wasseparated into 3 parts and pH of the reagents were 7.4, 7.2 and 6.7. Tosome of this reagent, 10 mM ATP (Amersham/Invitrogen) was added. The ATPwas serially diluted by quarter-logs from 10 mM to 0.32 mM.

Reactions were initiated by adding equal volumes (0.03 ml) of reagentand DMEM medium (Gibco/Invitrogen) containing 0.1% Prionex® (Pentapharm)and 1.4×10-5 mg/ml QUANTILUM® Luciferase (Promega Corporation).

Luminescence was measured for 0.5 seconds per well using Berthold Orionluminometer.

Results

The luciferase reaction using 5′-fluoroluciferin saturated with ATP at ahigher concentration than the reaction using luciferin (FIG. 56).Luciferase reactions containing 5′-fluoroluciferin could be used tomeasure ATP levels that exceed the linear range of reactions containingluciferin.

For the other fluoroluciferins, the intensity of luminescence increaseswith ATP (FIG. 57). Unexpectedly, the requirement for ATP is much higherin firefly luciferase reactions containing 7′-fluoroluciferin than inreactions containing luciferin where the K_(m) for ATP is usually below1 mM. 7′-fluorolucfierin could therefore be used in firefly luciferasereactions to quantitate ATP levels that exceed the linear range ofreactions utilizing luciferin.

The 5′-fluoroluciferin generated luminescence with λmax approximately 25nm blue-shifted from regular luciferin and 7′-fluoroluciferin generatedluminescence with λmax approximately 21 nm red-shifted from regularluciferin. The pH optima for the firefly luciferase reaction using7′-fluoroluciferin was approximately pH 7.5, much lower than luciferinwhere the pH optima is approximately 8.2 The firefly luciferase reactionalso maintained high comparative intensity at pH below 7.0.

Thus, 5′ fluoroluciferin has a lower pH optima (FIG. 59) and a spectralshift of about 10 nm more blue (FIG. 58) and 7′ fluoroluciferin haslower pH optima and a spectral shift of about 20 to 30 nm more red. Itwould be expected that 5′ fluoroaminoluciferin, 7′ fluoroaminoluciferinand 7′,7′ fluoroaminoluciferin, would have similar characteristics totheir luciferin counterpart.

Running reactions at lower pH increases stability of thiols andluciferin in a formulation, increasing overall reagent stability.Moreover, running reactions at lower pH may permit a coupled enzymereaction to generate higher light at low pH, and may permit a targetenzyme reaction to perform at a higher activity. Further, the reactionsmay result in higher luminescent intensities at lower pH (and may beused to reduce other enzyme activities, like ATPases or proteases). Inaddition, the spectral shift allows for better spectral separation inmultiplexing. For 7′ fluoroluciferin and 7′,7′ fluoroaminoluciferin, thespectral shift allows for increased measurable luminescence withPMT-based luminometers. 5′,7′ difluoroluciferin may have similarproperties. For 7′ fluoroluciferin, the requirement for ATP is very highfor the luminescence reaction and so it may be employed in reactionswhere there is a high Km for ATP without further reagent modification.

Summary

In summary, whereas light production from native luciferin changesdramatically over a pH range of pH 6.5 to 8.5, much less change was seenwith fluoroluciferin over these same pH values. This effect resulted innative luciferin generating more light at higher pH values butfluoroluciferin generating at least as much light if not more light atlower pH values. Therefore, those compounds are particularly usefulwhere extra signal is important in a reaction that must be performed atlowered pH values.

Moreover, some derivatives, including those with halogen modifications,can be utilized to generate light directly, and different derivativeswithin the broad scope of the luciferin derivatives of the invention,can vary in light production by several orders in magnitude

Luciferins modified with fluorine that have lower pH optima are usefulin reporter gene measurements, ATP-dependent assays, kinase assays, orother assays such as coupled assays, e.g., assays including caspaseassays such as caspase-3/7, caspase-8 or caspase-9 assays, or cathepsinB, cathepsin L or calpain assays, and as luciferase sensors, forexample, with derivatives that also have a protease site for a proteasewith an activity maxima lower than about pH 7.2.

Luciferins modified with fluorine that have a spectral shift of about 10nm or more blue are useful in all luminescent assays, and in particularin in situ or in vivo luminescent measurements (live cells in culture orlive animal imaging), reporter gene assays, and assays that useluciferase as a sensor.

Luciferins modified with fluorine that have a spectral shift of about 20to 30 nm or more red are useful to improve color quenching in 2 stepdual measurement assays, e.g., of a Renilla luciferase signal, or otherdual reporter gene assays, or in a multiplex assay for a nonluciferaseenzyme, such as a protease, with one or more reporter gene assays, or inother luminescent assays, such as in in situ or in vivo luminescentmeasurements (live cells in culture or live animal imaging), reportergene assays, and assays that use luciferase as a sensor.

Example 8 Luciferin Derivative for Beta-Gluronidase

Luciferin 6′-glucuronide was designed as a substrate forbeta-glucuronidase (GUS). The Km value of this compound in 50 mM MES, pH6.3, was 180+/−30 uM; this is reasonably similar to that of4-methylumbelliferyl glucuronide, a fluorescent substrate for GUS with aKm value of 59+/−8 uM. The luminescent substrate is more sensitive thanthe fluorescent substrate with a limit-of-detection of 0.28 ng/ml vs2.01 ng/ml, respectively. The source of the human enzyme wasSigma-Aldrich. GUS was incubated with the substrate in 50 mM MES, pH6.3, for 15 minutes. An equal volume of the P450-Glo luciferin detectionreagent was then added and the luminescence was measured at 2 minutes(Tables 51-52).

TABLE 51

[substrate] (uM) + GUS no GUS 2548.0 1,335,243 209,219 849.3 994,97791,975 283.1 696,778 34,127 94.4 423,619 13,091 31.5 232,533 5,068 10.5107,502 2,190 3.5 46,210 1,108 0.0 612 451

TABLE 52 signal-to-noise [GUS] (ng/ml) luminescence fluorescence 277.57300 861.4 138.8 3104 376.8 69.4 1411 164.6 34.7 649 76.5 17.3 296 34.78.7 133 15.6 4.3 68 7.5

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

What is claimed is:
 1. A compound according to formula VI:

wherein R¹ is —NR^(N1)R^(N2); wherein R^(N1) is H, C₃₋₈alkyl which maybe substituted with hydroxyl, halo, or amino, or —CH₂-A¹ or —CH₂-A²;R^(N2) is C₃₋₈alkyl which may be substituted with hydroxyl, halo, oramino or —CH₂-A¹ or —CH₂-A²; or A¹ is C₆₋₁₀aryl or C₆₋₁₀heteroaryl,which may be substituted with hydroxyl, halo, or amino; A² is C₆ aryl orC₆ heteroaryl, which may be substituted with hydroxyl, halo, or amino;and only one of R^(N1) or R^(N2) may be —CH₂-A¹; R⁶ is COR¹¹ wherein R¹¹is OH; R⁷ is H.
 2. A compound selected from the group consisting of